K. Beran

Demonstration of yeast scars by fluorescence microscopy

D URING the multiplication of yeast cells scars develop at cell-wall sites after separation of the daughter cell, appearing in the optical microscope as an unevenness of the surface. Barton [4] was the first to study systematically the types of scars in living, plasmolysed and stained cells of Saccharomyces cerevisiae. Later the structure of the cell wall in whole cells [3], in isolated membranes [l, 6, 10) and in ultrathin sections [I, 2, 91 was studied with the aid of the electron microscope. Since one can determine the relative age of cells from the number of scars, methods designed to demonstrate yeast bud scars should be of interest to cytologists [3], but hitherto there has been no method for studying scars on intact cells [5]. In the course of cytological studies of yeasts in this laboratory with aid of the fluorescence technique, the fluorochrome primulin was used among other agents. This non-toxic vital dye was used for the differentiation of viable and non-viable yeast cells [7, 8, 121. It was found, however, that at higher concentrations primulin causes an intense greenish fluorescence of the scars on the cell wall. The species studied were grown in modified medium of Olson and Johnson [ll] for 24-48 hr at 28°C on a shaker. The centrifuged and washed cells were suspended in the primulin dye solution in concentrations ranging from 1: 1000 to 1: 5000. Specimens were prepared from the suspension and the coverglass embedded in Vaseline. They were stained for 5-30 min. The specimens were observed in the Nf microscope (Zeiss) with an illuminator 01-17 (USSR) fixed on the microscope tubus. The source of illumination was a highpressure mercury vapour lamp DRSH-250. The following filters were used: FS-12 mm, exciter filter with 85 per cent transmission at 380 my, and the S3S-7, S3S-14, BS-8 filters and the ZHS-18 suppression filters. The photographs were made with the Miflex (Zeiss) camera, Ilford HPS film was used. Fluorescence microscopy was used for studying the yeast cell surface with the scars developing during budding, fission and reproduction by the intermediate process. For this purpose 28 species of various genera of yeasts were used. Budding yeasts.-In the majority of the strains investigated two kinds of scars were found, birth scars and bud scars, which have been previously described [l, 4, 61. The birth scar (Fig. la) indicates the site of the separation of the new cell from the mother cell. No fluorescent substance was found to accumulate at its margin. The bud scars (Fig. 1 b and 5) are formed after the separation of the daughter cells. On their margin a circular fluorescent thickening was observed so that these scars resemble a crater. In Saccharomyces cerevisiae the maximum of 25 bud scars were counted at the end of the logarithmic phase of growth.

The chitin-glucan complex ofSaccharomyces cerevisiae

The content of glucosamine in the walls of daughter (without bud scars) and mother (multiscar) cells ofSaccharomyces cerevisiae was examined in a control and after treatment with dilute alkali, acid and buffer. The occurrence of chitin in the bud and birth scars is discussed. The results of IR and X-ray analysis of cell-wall fractions indicate the presence of α-chitin which is a part of the chitin-glucan complex. The size of the crystallite of α-chitin in this complex is about 60 Å.

Types of multiplication scars in yeasts, demonstrated by fluorescence microscopy.

A new method permitting the study of scars on intact cells of different species of yeasts by means of secondary primulin fluorescence is described. The existence of two types of scars in budding yeasts was confirmed and their morphology was described in intact cells. In fission yeasts a number of division scars was found in individual cells and changes occurring in the lateral walls as a result of cytokinetic process were observed. In yeasts reproducing by bipolar budding, a new type of scar—the multiple scar—with an important morphogenetic function was discovered. The possibilities and prospects of the new method are discussed.AbstractОписЫвается нояій метод изучения шрамов на нена рушеннЫх клетках различнЫх видов дрожжей на основании вторичной флуоресценции примулина. БЫло подтверждено наличие у почкующихся дрожжей двух видов шрамов. ОписЫвается морфология шрамов ненарушеннЫх клеток. У делящихся дрожжей бЫло обнаружено большое количество шрамов от деления (division scars) на отделънЫх клетках; наблюдалисъ изменения боковЫх ктенок, развивающиеся в резулътате цитокинетического процесса. У дрожжей с бинолярнЫм способом бегетативнобо размножения. бЫл обнаружен новЫй тип шрамов—множественнЫе шрамЫ (multiple scars), связаннЫе с важной мовфогенетической функцией.—Обсуждаются возможности и перспективЫ нового метода.

The glucan-chitin complex in Saccharomyces cerevisiae

Scar rings (SR) from scarless cells at the early stages of budding and mature bud scars from Saccharomyces cerevisiae isolated by both chemical and enzymic treatment of cell walls were observed by selected-area electron diffraction (SAED), X-ray diffraction and electron microscopy with simultaneous physico-chemical characterization (including molar mass, intrinsic viscosity and crystallite size) of α-chitin and glucan. The SR, composed of glucan with strong 0.608; 0.397 and 0.293 nm X-ray reflections, was formed at the start of budding. The SAED patterns of α-chitin both in the adjacent circular zone and in the parts of newly formed primary septum (PS), observed when the development of the PS started, did not differ from those of α-chitin in the single mature bud scar. The bud scar consisted of α-chitin, glucan and mannan and their content, as well as the crystallite size of chitin, depended on the mode of preparation.

The chitin-glucan complex of Saccharomyces cerevisiae. II. Location of the complex in the encircling region of the bud sear.

Fractions of the cell wall ofSaccharomyces cerevisiae where a α-chtin-glucan composition was established, were examined under the electron microscope as well as with phase fluorescence. The results indicate that the α-chitin-glucan complex shows a fibrillar arrangement and is localized in the socalled encircling region of the bud scar which has a tear-like appearance and is electron-transparent in ultrathin sections. There are indications of the presence of chitin even in the primary septum.

Fractionation of a population ofSaccharomyces cerevisiae yeasts by centrifugation in a dextran gradient

A method of the fractionation of aSaccharomyces cerevisiae yeast population in dextran gradients is described. The elaboration of this method was based on the finding of a correlation between the size of individual cells and the number of bud scars on their surface and rapid indication of the scars by fluorescence microscopy. The basic conditions for fractionation (determined experimentally) were as follows: 2 ml. yeast suspension (100 mg. dry weight) was applied to the surface of a continuous dextran gradient of 9–16% concentration and was centrifuged at a relative centrifugal force of 200 G for 15 minutes. In fractionation of a whole population, the best fractionation was obtained in a linear gradient. Repeated separation of fractions obtained by centrifugation in a linear gradient in a concave gradient further separated cells without bud scars and accumulated cells with five scars and over. Three fractions were obtained by this technique. The first contained 90–98% cells without bud scars, the second 55–65% cells with 1–4 bud scars and the third 50% cells with five bud scars and over.AbstractОписан метод фракционирования популяции дрожжей Saccharomyces cerevisiae в декстрановых градиентах. Основной предпосылкой для разработки этого метода было обнаружение отношения между размерами отдельных клеток и количеством материнских шрамов на их поверхности, а также способ быстрой индикации шрамов с помощью флуоресцентной микроскопии. Экспериментально были установлены следующие основные условия для фракционирования: суспензию дрожжей (100 мг сухого вещества) в 2 мл вносили в горизонт декстрана с концентрацией, постепенно и непрерывно повышающейся от 9 до 16%, и центрифугировали 15 мин. при относительной центробежной силе 200 G. При центрифугировании всей популяции наиболее успешное фракционирование было получено в линейном градиенте. Путем дальнейшего разделения фракций, полученных в резулятате центрифугирования в линейном градиенте, с применением вогнутого градиента удалось отделить еще клетки без материнских шрамов и скопления клеток с 5 и более шрамами. Так были получены 3 фракции: первая содержала 90–98% клеток без материнских шрамов, вторая— 55–65% клеток c 1–4 материнскими шрамами, а третья—50% клеток с 5 и более материнскими шрамами.

Activation of proteolytic enzymes during autolysis of disintegrated baker’s Yeast

Disintegration substantially acclerates autolysis of yeast cells. Three proteases (A, B, and C) take part in the autolytic process, protease A being the activator of the other two enzymes. The role of proteases B and C in the process depends on temperature. At 40 °C both proteases are active while at 50 °C the major role is played by protease C. At 40 °C NaCl acts as inhibitor while at 50 °C it activates the process.

The chitin-glucan complex of Saccharomyces cerevisiae. III. Electron-microscopic study of the prebudding stage.

Differentiation of the cell wall of Saccharomyces cerevisiae at the site of the future bud was followed. A lentil-like structure originates on the inner side of the cell wall during the first phase. At the same time, an electron-dense layer occurs at the boundary between the inner layer of the cell wall and the lentil-like structure. During the second phase granular material is accumulated at the lower side of the lentil-like structure. During the third phase the lentil-like structure is split apart due to proliferation of the granular material resulting in formation of the base of the encircling region. The marked electron-dense layer observed from the first phase is attached to the surface of the encircling region during differentiation of the latter. During the budding proper the outer layers of the cell wall protrude and the end of the encircling region, together with the adjacent electron-dense layer, acquire their definitive appearance of rings, observed as marked electron-transparent and electron-dense tears on ultrathin sections.Differentiation of the cell wall ofSaccharomyces cerevisiae at the site of the future bud was followed. A lentil-like structure originates on the inner side of the cell wall during the first phase. At the same time, an electron-dense layer occurs at the boundary between the inner layer of the cell wall and the lentil-like structure. During the second phase granular material is accumulated at the lower side of the lentil-like structure. During the third phase the lentil-like structure is split apart due to proliferation of the granular material resulting in formation of the base of the encircling region. The marked electron-dense layer observed from the first phase is attached to the surface of the encircling region during differentiation of the latter. During the budding proper the outer layers of the cell wall protrude and the end of the encircling region, together with the adjacent electron-dense layer, acquire their definitive appearance of rings, observed as marked electron-transparent and electron-dense tears on ultrathin sections.

Fed-Batch cultivation ofSaccharomyces cerevisiae with increased content of Δ5,7-sterols

Cultivation ofSaccharomyces cerevisiae with a linear nutrient feed was used to test the validity of a mathematical model describing changes in the physiological state of the culture. Markers of these changes were the concentrations of proteins and ‡5,7-sterols in yeast dry mass. The model was used to optimize the production of these sterols with regard to the magnitude and composition of nutrient feed.

Ergosterol synthesis and population Analysis of a fed-batch fermentation inSaccharomyces cerevisiae

Saccharomyces cerevisiae with an increased content of ergosterol or Δ5,7-sterols, growing on a molasses medium with a feed of ethanol and (NH4)2HPO4, was analyzed as to the age of cell population. The analysis was done by centrifugation in a dextran gradient and by a fluorescence-microscopic technique. In the phase of batch fermentation at a mean specific growth rate of 0.22 h-1 daughter cells contained less than 1% ergosterol while the ergosterol content of mother cells depended on the time of cultivation, a maximum level (4 %) being found after two generation times. In the fed-batch phase at a mean growth rate of 0.052 h-1, both daughter and mother cells contained about the same amount of ergosterol (4.7–3.5 %). Differences between daughter and mother cells are discussed in view of the relationship between the growth rate and the growth cycle.