Budding Cycle Research Articles

The cryptic beta-fructofuranosidase of Saccharomyces rouxii.

The synthesis of beta-fructofuranosidase in synchronously dividing cells of S. rouxii was continuous (as opposed to periodic) throughout the budding cycle and followed the increase in cell mass. Similar patterns for cell mass and enzyme increases were observed even in phosphate-deprived cells which did not divide. The beta-fructofuranosidase activity remained physically cryptic throughout the cell cycle as evidenced by analyses on equilibrium density gradient fractions. The beta-fructofuranosidase activity released from mechanically disrupted cells resisted sedimentation when subjected to 131 000 g for 1 h, thus ruling out membrane association. Ethyl acetate was routinely employed to break the crypticity barrier. Enzyme in cell-free extract or in cells was equally sensitive to inactivation at pH values below 5 in the presence of ethyl acetate, which suggested that this is an inherent property of the enzyme in question and not a reflection of proteolytic inactivation. The status of beta-fructofuranosidase in selected species of Saccharomyces was compared with that for S. rouxii and a close similarity with S. bisporus var. mellis was noted. The degree of crypticity encountered in genetically defined strains of S. cerevisiae (e.g. X2180 a/alpha) was relatively high (42%) compared with that for commercially derived bakers' and brewers' strains (about 6%). Extant data on the cryptic beta-fructofuranosidase of S. rouxii are evaluated and the utility of this system for studying enzyme translocation is discussed.

Microtubule function and its relation to cellular development and the yeast cell cycle in Wangiella dermatitidis.

The microtubule inhibitor nocodazole (methyl-5-[2-(thienylcarbonyl)-1H-benzimidazol-2-yl]-carbamate) prevented nuclear migration and nuclear division in yeasts and developing multicellular forms of the polymorphic fungus Wangiella dermatitidis. It did not prevent yeast bud formation during at least two or three budding cycles, and caused yeasts to accumulate as premitotic forms with one to three buds. The effects of the drug suggested that at least three control pathways were involved in the yeast cell cycle; that the nocodazole block point was separate from the execution points of two temperature-sensitive mutations which lead to multicellularity; and that microtubules were controlling neither the yeast budding process nor the development of multicellular forms.

Eeffect of pH on growth in a respiration‐deficient mutant of Saccharomyces cerevisiae

AbstractThe effects of pH on the budding cycle of a respiration‐deficient mutant of Saccharomyces cerevisiae were investigation by monitoring the time course of bud development on single cells. The volume of each bud was measured at various time intervals between the inception of its development and inception of development of the next bud on the mother cell. A previous report that the budding cycle consisted of two phases (a rapid‐growth phase and a slow‐growth phase) was confirmed. With increase in pH from 3.8 to 6.0 the budding cycle shortened as a result of both increase in rate of the rapid‐growth phase and decrease in the duration of the slow‐growth phase. Although further increase in pH to 7.4 further increased the rate of the rapid‐growth phase, the budding cycle lengthened as a result of an increase in time lag and increase in duration of the slow‐growth phase. The growth rate, in terms of bud volume, conformed with the expression: (1/V)(dV/dτ) = ξ exp(–V/η), where the values of ξ and η were dependent on pH. The cell volume distribution in a batch culture was compared with the cell volume distribution calculated from the growth curve of a single bud. Similarities in the curves suggested that the growth pattern of a whole culture reflected the growth pattern of a single cell.

Life cycle of a budding and appendaged bacterium belonging to morphotype IV of theBlastocaulis-Planctomyces group

The budding process of cell division in a bacterial strain (ICPB 4232) belonging to morphotype IV of theBlastocaulis-Planctomyces group was followed in aerobic slide cultures. Under the conditions employed, 3 h elapsed from the appearance of the earliest discernible stage of daughter cell formation to the separation of the mature ovoid bud. Budding invariably occurred at the broader pole of the ovoid, nonmotile mother cell. The separated daughter cell (bud), a motile swarmer, locomoted by means of an ensheathed flagellum attached at its broader pole. Approximately 30 h were required for the swarmer to develop into a sessile mother cell and initiate a new budding cycle. A mother cell was capable of producing an individual bud repeatedly at the same budding site. Transmission electron microscopy was employed to determine ultrastructural details regarding the single ensheathed flagellum, the polarly distributed crateriform structures and numerous pili, the excreted holdfast, the multifibrillar major appendage (a fascicle), and daughter cell (bud) formation.

Effects of the antimicrotubular cancerostatic drug nocodazole on the yeast Saccharomyces cerevisiae

Nocodazole completely inhibited cell division of Saccharomyces cerevisiae, contrary to methyl-benzimidazole-2-ylcarbamate, estimated by cell counting. Growth as measured by turbidity and dry weight estimations, however, was not influenced. Treatment for two hours with nocodazole interrupts the budding cycle of the yeast within a period when bud and mother cells have reached equal sizes. Dependent on duration of nocodazole treatment stretched or dumb-bell shaped nuclei are localized between bud and mother cell or two daughter nuclei are present. It has been shown by electron microscopy that nocodazole neither destroyed karyo- nor ground-plasmic microtubules. The spindle pole bodies (spb), however, reached double the normal size approximately.

Effects of low concentrations of zinc on the growth and dimorphism of Candida albicans: evidence for zinc-resistant and -sensitive pathways for mycelium formation

In this analysis we have examined in detail the effects of low concentrations of zinc on the growth and dimorphism of Candida albicans. Evidence is presented that micromolar concentrations of zinc added to growth cultures grown at 25 degrees C (i) cause a twofold increase in the final concentration of spheres at sationary phase, (ii) result in an asynchronous block in the budding cycle at stationary phase, (iii) completely suppress mycelium formation in two independently isolated human strains which produce low but significant levels of mycelia at stationary phase, and (iv) completely suppress mycelium formation in cultures of mutant M10, in which over 60% of the cells form mycelia at stationary phase. In contrast, micromolar concentrations of zinc do not inhibit mycelium formation induced by releasing cells from stationary-phase cultures into fresh medium at 37 degrees C. In addition, if zinc is present in the growth medium of the initial culture at 25 degrees C, the average time of subsequent mycelium formation after release into fresh medium at 37 degrees C is halved. It is demonstrated that the above effects are specific to zinc. The possibility of alterante pathways for mycelium formation is suggested, and the medical implications of this possibility are discussed.

Chitin structures of the cell walls of synchronously grown virgin cells ofSaccharomyces cerevisiae

The ability of a lytic beta-glucanase of Arthrobacter GJM-1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin-containing fraction was employed for the isolation of chitin-rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin-rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.

Chitin structures of the cell walls of synchronously grown virgin cells of Saccharomyces cerevisiae

AbstractThe ability of a lytic β‐glucanase of Arthrobacter GJM‐1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin‐containing fraction was employed for the isolation of chitin‐rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin‐rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.

The Uptake and Incorporation of 14C-labelled Sugars into Polysaccharides during Wort Fermentation by a Synchronous Culture of Brewer's Yeast

SUMMARY: The incorporation of [U-14C]glucose, -maltose and -maltotriose into the polysaccharides of a brewer's yeast, Saccharomyces cerevisiae (NCYC240), in synchronous culture was followed during the fermentation of brewer's wort. The synchrony was induced by repeated subculture in brewer's wort at regular intervals, using cells skimmed from the surface of previous brews. The 14C-labelled sugars were assimilated simultaneously by the yeast and small but significant amounts of each incorporated into cellular polysaccharides. The low molecular weight acid-soluble fraction exhibited a cyclic fluctuation attributable to changes in enzymic activity associated with the synchronous behaviour of the yeast. Cyclic changes were also observed in the incorporation of labelled sugars into the mannan fraction of the walls, the uptake rising to successive maxima at the same stage of each budding cycle, followed by a rapid loss as the next cycle began. The incorporation of 14C into glycogen exhibited a stepwise character, in which brief periods of glycogen buildup alternated with longer periods of stability. The periods of glycogen biosynthesis coincided with the periods of most intensive budding. Incorporation of 14C into the glucan of the walls was a continuous process and could not be correlated with any particular phase of the cell cycle. Incorporation of 14C into glycogen and glucan was stable.

Localization of free sulphydryl groups by ferricyanide reduction in developing conidiophores of Aspergillus nidulans.

Light and electron microscope observations were made on colonies ofAspergillus nidulans treated before fixation with the ferricyanide reaction of Chevremont & Frederic. Sulphydryl groups, blockable by maleimide, were found at the conidiophore surface, internal to the wall, especially at the septa. Maleimide-stable reactivity, and at first some sulphydryl reactivity, was associated with the pigmenting regions of the exterior of the wall. No reactivity was found at any growing apex and none on the cell surface until the end of a budding cycle. The free sulphydryl groups were, therefore, associated with cell surface activity in the maturing cell immediately prior to pigmentation.

Recovery of yeast from transient inhibition of DNA synthesis.

IN the simple eukaryotic cell Saccharomyces cerevisiae nuclear division, cell division and the start of new budding cycles depend on DNA synthesis1. Protein synthesis is required for the recovery of new budding and cell division after transient exposure to hydroxyurea, a specific reversible inhibitor of DNA synthesis2. In this report I have attempted to answer two questions. (1) Does recovery of nuclear division require protein synthesis? (2) Does recovery of DNA synthesis itself require protein synthesis?

Inactivation of chitin synthase in Saccharomyces cerevisiae.

The activity of chitin synthase extracted from whole cells of Saccharomyces cerevisiae shows reproducible changes during the course of batch cultivation. During exponential growth 5–10% of the enzyme occurs in the active form, whereas in the stationary phase no active enzyme can be detected. Of three yeast proteinases, A, B and C, only B is able to activate pre-chitin synthase and inactivate chitin synthase. A new model of the regulation is presented which accounts for the specific location as well as for termination of chitin synthesis during the budding cycle.

Regulation of carbohydrate composition of Saccharomyces cerevisiae under growth limitation.

1. The carbohydrate composition of Saccharomyces cerevisiae cultivated aerobically in the chemostat at different growth rates was investigated. It was compared with the specific activities of enzymes of the reserve carbohydrate pathways. Under glucose limitation the synthesis of reserve carbohydrates increases strongly with increasing limitation of substrate supply, i.e., decreasing growth rate. Fermenting cells accumulate only glycogen, whereas more slowly growing organism with a purely oxidative metabolism contain trehalose and high amounts of glycogen. 2. During the budding process the purely respirative cells degrade part of their reserves. In the presence of excess substrate the reserve carbohydrate content is low and remains unchanged during the cell cycle. 3. The percentage of structural carbohydrates in the cells shows little changes during the budding cycle, being scarcely influence by the growth rate. 4. At a growth rate of 0.077 h-1 the cells cultivated under glucose limitation contain even a higher amount of reserves than those grown under nitrogen limitation. 5. The course of the specific activities of UDP-glucose pyrophosphorylase and glycogen phosphorylase as a function of the growth rate follows that of the reserve carbohydrates.

Observations on budding cells of Torulopsis glabrata.

The diminutive pathogenic yeast Torulopsis glabrata was studied by light and electron microscopy, with special attention to the budding process. Polysaccharides were also extracted to determine glycogen in cytoplasmic particles. Elliptical single cells, with a mean volume of 13 μm3, possess ultrastructural features common in other yeasts, viz. nucleus with double membrane and pores, nucleolus, endoplasmic reticulum, mitochondria, vacuoles, lipid bodies, ribosomes, tripartite plasma membrane, and fibrillar wall with outer electron-dense layer. Numerous pocket-like invaginations of varying size and orientation are formed by the plasma membrane. Buds seem to be preceded by an elaboration of such invaginations and by tubular profiles of the endoplasmic reticulum present in the area of the emerging bud. As the bud protrudes, it appears bounded by a layer of newly synthesized wall material. In general, this process is similar to that in other yeasts (e.g., Saccharomyces) but no vesicles were observed in the peripheral cytoplasm of the mother cell during the initial stages of budding. Various stages in the budding cycle are illustrated.

Isolation of glucanase-containing particles from budding Saccharomyces cerevisiae.

In an investigation of the role of glucanases in modifying yeast cell walls at the location of new buds, vesicles derived from the endoplasmic reticulum, which are secreted locally into the cell wall of growing buds and may be involved in the secretion of glucanases, have been isolated.In yeast, exo-beta-1,3-glucanase (EC 3.2.1.6) is present both extra- and intracellularly. Exponentially growing cells contain about 16% of the enzyme activity intracellularly (within the plasmalemma). Most, if not all, of this intracellular glucanase is sedimentable. Of the three classes of subcellular particles that contain glucanase, one is almost completely absent from stationary phase cells and almost absent from cells of the late budding phase of the budding cycle. These particles were isolated from budding cells by combined differential and density gradient centrifugation. They contain exo- and endo-beta-1,3-glucanases, mannan, and protein. The isolate consists mainly of membrane-bounded particles with diameters corresponding to those of the secretory vesicles observed in situ. It is concluded that these particles are identical with the vesicles derived from the endoplasmic reticulum.

Vacuolar dynamics in synchronously budding yeast

A simple and rapid method for obtaining synchronously budding cultures of Saccharomyces cerevisiae is described. Synchronous cultures were started with homogeneous cell fractions isolated from exponentially growing cultures by isopycnic centrifugation in osmotically inactive media. The technique of fractionation is based on changes of cell density throughout the budding cycle. These changes are correlated with vacuolar changes observed in the light and electron microscope. During bud initiation the large vacuoles in late budding cells shrink and fragment into small vacuoles. Simultaneously the density of the cells increases. Later stages of the budding cycle are characterized by the distribution of the small vacuoles between mother and daughter cell, followed by their fusion and expansion, and by a decreasing density of the cells. The relative changes in cell density and dry weight and in the content of different macromolecules during the budding cycle suggest a cyclic change between utilization of endogenous and exogenous substrates. This is discussed in terms of a cyclic consumption and accumulation of vacuolar pools.

Changes in carbohydrate composition and trehalase-activity during the budding cycle of Saccharomyces cerevisiae.

The carbohydrate composition and the specific activity of the trehalase of cyclic partially synchronised yeast populations have been investigated. Under glucose limitation and appropriate cultural conditions synchronous growth in a chemostat was achieved. The cells accumulated the reserve carbohydrates during the single cell phase between two buddings. The rapid degradation of part of these reserves began shortly before the swelling of the bud. The importance of the mobilisation of endogenous reserves for the development of the cell is discussed. The specific activity of the trehalase changed during the budding cycle. The result gives rise to the assumption that the synthesis of this enzyme is linked to the growth cycle.

Energetics of the budding cycle of Saccharomyces cerevisiae during glucose limited aerobic growth.

A method for the estimation of the yield on energy (YATP) and of the efficiency of oxidative phosphorylation, in vivo (P/O ratio) is described, which is based on the measurement of effective gas exchange values (\(Q_{O_2 }\) and \(Q_{CO_2 }\)) and of the yield coefficient Y of continuously growing populations of baker's yeast which vary in the degree of fermentation and respiration. For YATP a value of 12.0±0.5 \(\frac{{{\text{mg dry weight formed}}}}{{{\text{mMole ATP}}}}\) and for P/O ratio one of 1.1±0.05 \(\frac{{{\text{mMole ATP}}}}{{{\text{1/2 mMole O}}_{\text{2}} }}\) was found and seems to be independent of the type of glucose catabolism (under glucose limitation).

Enzyme pattern and aerobic growth of Saccharomyces cerevisiae under various degrees of glucose limitation.

The enzyme pattern of Saccharomyces cerevisiae was followed during batch growth and in continuous culture in a synthetic medium limited for glucose under aerobic conditions. Seven enzymes were measured: succinate-cytochrome c oxidoreductase, malate dehydrogenase, nicotinamide adenine dinucleotide-linked glutamate dehydrogenase, malate synthase, isocitrate lyase, aldolase, and nicotinamide adenine dinucleotide phosphate (NADP(+))-linked glutamate dehydrogenase. During fermentation of glucose and high growth rate (mu) during the first log phase in batch experiments, the first five enzymes (group I) were repressed, and aldolase and NADP(+)-linked glutamate dehydrogenase (group II) were derepressed. During growth on the accumulated ethyl alcohol and lower mu, the group I enzymes were preferentially formed and the other two were repressed. A sequence of derepression of the group I enzymes was found during the shift from glucose to ethyl alcohol metabolism, which can be correlated with a strong increase in the percentage of single (nonbudding) cells in the population. A correlation between the state of cells in the budding cycle and enzyme repression and derepression is suggested. In continuous culture, the enzyme pattern was shown to be related to the growth rate. The group I enzymes were repressed at high growth rates, while the group II enzymes were derepressed. Each enzyme exhibits a different dependence. The enzyme pattern is shown to depend on the rate of substrate consumption as well as on the type of metabolism and to be correlated with the budding cycle. The enzyme pattern is considered to be controlled by changes of intracellular catabolic or metabolic conditions inherent in the division cycle.

Adaptation of Yeast to Cadmium

By using the method of sib selection followed by the replica-plating, a cadmium resistant clone (r1) was isolated from a sensitive strain (Par), and a clone of a higher resistance level (r2) from r1. Thus, r1 and r2 were shown to be of pre-adaptive nature. However, the strain of still higher resistance, obtainable by culturing in the presence of cadmium, has not been isolated by the indirect selection.The mutation rate from Par to r1 and that from r1 to r2 were 6.3×10-9 and 4.0× 10-8 per cell per budding cycle, respectively. Growth of r1 was slower than Par, and r2 than r1, in their mixed cultures.r1 was more resistant to copper, cobalt and nickel than Par, but r2 was less so than r1 except for nickel.