Commitment to germ tube or bud formation during release from stationary phase in Candida albicans.

Cells of the pathogenic yeast Candida albicans accumulate as unbudded singlets at stationary phase in defined medium at 25 °C. When released into fresh medium at 37 °C and pH 6.5, these cells will synchronously form elongate pseudomycelia, and when released into fresh medium at either 25 °C, pH 6.5, or 37 °C, pH 4.5, they will synchronously form buds. Using pH and temperature shift experiments, we have examined when cells become committed to pseudomycelium formation and bud formation under conditions conducive to each growth form respectively. It is demonstrated that in either case commitment occurs long after release from stationary phase, at approximately the same time the first evagination is visible on the cell's surface. In addition, it is demonstrated that once a released cell has formed a bud, it and its progeny lose the capacity to form pseudomycelia until they re-enter stationary phase; on the other hand, elongating pseudomycelia retain the capacity to form buds. The possible relationships of the commitment events to septation and to the cell cycle are discussed.     

A characterization of pH-regulated dimorphism in Candida albicans

When cells of the dimorphic yeast Candida albicans are grown to stationary phase in defined liquid medium at 25°C, they accumulate as singlets in G_l of the cell cycle. When these pluripotent, stationary phase singlets are released into fresh medium at 37°C, they synchronously evaginate after an average period of 135 to 140 minutes and form either buds or mycelia, depending upon the pH of the medium into which they are released. This method of dimorphic regulation offers the distinct advantage of comparability and serves as a very precise method for temporal comparisons of molecular and cytological events related to the establishment of the alternate growth phenotypes. In the present report, we have carefully examined the effects of individually varying pH or temperature on the length of the pre-evagination period, the population synchrony for evagination, and the phenotype of daughter cells. Exact phenotypic transition points, optima, and upper limits are defined for both temperature and pH. In addition, a method of pH-regulated dimorphism is developed in which the original temperature shift is removed from the inductive process. Finally, a common transition phenotype is described for cells reverting from the initial mycelial to budding phenotype when either pH or temperature traverse their respective transition points. The advantages as well as limitations of pH-regulated dimorphism are discussed in detail.     

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.     

Regulation of cell size in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.     

Filament ring formation in the dimorphic yeast Candida albicans

Stationary phase cultures of Candida albicans inoculated into fresh medium at 37 degrees C synchronously from buds at pH 4.5 and mycelia at pH 6.5. During bud formation, a filament ring forms just under the plasma membrane at the mother cell-bud junction at roughly the time of evagination. A filament ring also forms in mycelium-forming cells, but it appears later than in a budding cell and it is positioned along the elongating mycelium, on the average 2 microns from the mother cell-mycelium junction. Sections of filament rings in early and late budding cells and in mycelia appear similar. Each contains approximately 11 to 12 filaments equidistant from one another and closely associated with the plasma membrane. In both budding and mycelium-forming cells, the filament ring disappears when the primary septum grows inward. The close temporal and spatial association of the filament ring and the subsequent chitin-containing septum suggests a role for the filament ring in septum formation. In addition, a close temporal correlation is demonstrated between filament ring formation and the time at which cells become committed to bud formation at pH 4.5 and mycelium formation at pH 6.5. The temporal and spatial differences in filament ring formation between the two growth forms also suggest a simple model for the positioning of the filament ring.     

Differences in actin localization during bud and hypha formation in the yeast Candida albicans

Stationary phase cells of Candida albicans can form either a bud or a hypha, depending upon the pH of the medium into which they are released. At low pH, cells form an ellipsoidal bud and at high pH, cells form an elongated hypha. By staining cells with rhodamine-conjugated phalloidin, we have compared the dynamics of actin localization during the formation of buds and hyphae. Before evagination, actin granules were distributed throughout the cytoplasmic cortex in both budding and hypha-forming cells. Just before evagination, actin granules clustered at the site of evagination, then filled the early evagination in both budding and hypha-forming cells. With continued bud growth, the actin granules then redistributed throughout the cytoplasmic cortex. In marked contrast, with continued hyphal growth, the majority of actin granules clustered at the hyphal apex. This distinct difference in actin granule localization may be related to the distinct differences in the expansion zones of the cell wall recently demonstrated between growing buds and hyphae. The spatial and temporal dynamics of the large neck actin granules and of actin fibres are also described.     

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

The 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.     

The temporal regulation of protein synthesis during synchronous bud or mycelium formation in the dimorphic yeast Candida albicans

When stationary phase cells of the dimorphic yeast Candida albicans are diluted into fresh medium at 37°C at either pH 4.5 or pH 6.5, they evaginate at exactly the same time and with the same synchrony. However, they then grow in the budding yeast form at the former pH and in the elongate mycelium form at the latter pH. Three phases of protein synthesis are distinguished for cells forming either buds or mycelia: an initial 50-min period (phase I) during which total cell protein remains constant and the rate of incorporation of labeled amino acid into protein is virtually zero; a second period (phase II) during which there is a slow but constant increase in both total cell protein and the rate of incorporation; and a third period (phase III) during which there is a dramatic increase in both total cell protein and the rate of incorporation. The transition from phase I to phase II occurs at the same time for cells forming either buds or mycelia, but the transition from phase II to phase III occurs 20 to 30 min later in the mycelium than in the bud forming population, the same temporal difference observed for phenotypic commitment. The polypeptides synthesized during phases II and III were first analyzed by one-dimensional polyacrylamide gel electrophoresis. The patterns are similar for the two phenotypes. The major polypeptides synthesized during phase II are also synthesized during phase III, but in addition, a group of at least four new major polypeptides appear during phase III for both phenotypes. The minor polypeptides synthesized during phase III were also compared between the two phenotypes by two-dimensional polyacrylamide gel electrophoresis. The patterns, including roughly 200 distinguishable polypeptides, were similar. The similarities in the patterns of protein synthesis and the delay in the onset of phase III in mycelium forming cells are discussed in terms of phenotypic commitment. From these considerations, alternate hypotheses for the regulation of fungal dimorphism, in particular, and cell divergence, in general, are proposed.     

Ultrastructure of Saccharomyces cerevisiae strain AG1-7 and its responses to changes in environment

Asynchronous populations of the budding yeast Saccharomyces cerevisiae strain AG1-7 were examined by freeze-fracture electron microscopy for ultrastructural changes occurring in response to changes in the environment, specifically the following: temperature (23 or 37 degrees C); cell density (exponential, early stationary, and stationary phases); various periods of nitrogen starvation at low cell density, and return of nitrogen-starved cells to nitrogen-replete medium. This information has been gathered in preparation for ultrastructural examination of comparable responses of temperature-sensitive cell-cycle mutants. The plasma membrane was found to be particularly responsive to changes in environment. A high proportion (75%) of cells in exponential phase populations at 37 degrees C displayed paracrystalline arrays of plasma membrane particles, whereas this proportion was much lower (20%) at 23 degrees C in the same medium; plasma membrane grooves were longer at 37 than at 23 degrees C. In budded cells, the mother cell displayed paracrystalline arrays more frequently than the bud. Entry of cells into stationary phase, either through permitting population growth or by limiting nitrogen supply, resulted in increases in numbers of paracrystalline arrays and grooves. Groove depth also increased. The paracrystalline-array and groove-density responses were independent, both during entry into stationary phase and during the subsequent lag phase. Unusual groove forms appeared during stationary phase in high cell density populations, but not in low cell density nitrogen-starved populations. "Aggregate" and "geometric" tonoplast forms, previously described in strain A364A when grown under some of the conditions used here, were not found in AG1-7 under any of the conditions used here. It was demonstrated that particle-free patches can arise rapidly on the tonoplast of AG1-7 in response to temperature change from 37 to 23 degrees C. During stationary phase, spherosomes (lipid droplets) increased in size, particularly in response to nitrogen depletion. After 72 h of nitrogen starvation, about 10% of cell volume consisted of spherosomes. Changes in vacuolar content and mitochondrial form were also noted during entry into stationary phase.     

The dependency of nuclear division on volume in the dimorphic yeast Candida albicans

When stationary phase cells of the dimorphic yeast Candida albicans are induced to synchronously form mycelia, over 90% of the cells undergo nuclear division. However, when stationary phase cells are induced to synchronously form buds, less than half undergo nuclear division even though all form buds. The majority of cells which do not undergo nuclear division form buds with volumes below a threshold value and the majority of cells which do undergo nuclear division form buds with volumes above this threshold value. In this report, we have investigated the possibilities that cells which form small buds do not attain a particular mass threshold. Cell cultures were examined for DNA replication, dry weight, and protein content during synchronous bud and during synchronous mycelium formation. Evidence is presented which indicates that the lack of nuclear division in over half of a budding population is due to low daughter cell volumes or to low surface areas, and not to their failure to attain a mass threshold or to replicate their DNA. The dependency of nuclear division on daughter cell volume is discussed.     

The involvement of cell wall expansion in the two modes of mycelium formation of Candida albicans

When budding cells of Candida albicans are starved for 20 min and then diluted into fresh nutrient medium at 37 degrees C, pH 6.7, they form mycelia by two alternative modes. For cells with small buds, the bud expands apically, resulting in a transiently tapered daughter cell. With continued growth, the daughter cell tapers into an elongated mycelium. For cells with large buds, the bud completes expansion in the budding form, the mother cell and then the daughter bud evaginate, and the evaginations grow as mycelia. The present study investigates whether the temporal and spatial changes in the zones of wall expansion during bud growth are involved in the two modes of mycelium formation. Data are presented which demonstrate that the transition circumference which determines the two modes of mycelium formation and the transition circumference at which the active apical expansion zone shuts down are both 7 micron. This exact correlation suggests that starved cells with buds with a circumference of less than 7 micron form mycelia in the tapering mode due to the reactivation of the still present apical expansion zone, and that starved cells with buds with a circumference greater than 7 micron complete bud growth by general expansion due to the absence of the apical expansion zone at the time of starvation.     

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5–8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.     

The programs of protein synthesis accompanying the establishment of alternative phenotypes in Candida albicans

Under the regime of pH-regulated dimorphism, stationary phase cells of the dimorphic yeast Candida albicans can be induced to form exclusively and synchronously ellipsoidal buds or elongate mycelia at the same temperature and in the same nutrient medium, the sole determinant of phenotype in this case being pH. Employing pH-regulated dimorphism, cells were pulse-labeled with [³⁵S]-methionine during three consecutive intervals encompassing the preevagination period, the period including evagination and phenotypic commitment, and the post-evagination period. Labeled polypeptides were analyzed by 2D-PAGE. Of the 374 polypeptides examined, the majority (237) did not differ significantly in relative incorporation between the three pulse periods and were similar between budding and mycelium-forming populations. Sixty polypeptides were labeled at negligible or relatively low levels during the first pulse period, but at significantly higher levels during the second and third or third pulse periods. All but one were similar between budding and mycelium-forming populations. Seventeen polypeptides were synthesized at relatively high levels during the first pulse period, but at reduced or negligible levels during the second and third or third pulse periods. All but one were similar between budding and mycelium-forming populations. Only two polypeptides were found to be associated exclusively with mycelium-forming cultures, two associated exclusively with budding cultures, and two enriched significantly in budding cultures of wild-type cells. Employing a variant, MD20, which forms buds at both low and high pH, it was demonstrated that only one mycelium-associated polypeptide and only one bud-associated polypeptide are phenotype rather than pH-specific. Limits to this method of phenotype comparison are outlined, and the unusual similarity rather than dissimilarity in the programs of gene expression between the diverging populations considered in terms of phenotypic regulation.     

Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.     

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

Behaviour ofPropionibacterium shermanii in alkaline medium

Physiological and morphological properties ofPropionibacterium shermanii were estimated in a liquid semisynthetic medium of initial pH between 7 – 10. Prolongation of the lag phase and a shift of the stationary phase occurred at higher initial pH values of the medium, The growth response of the strain depended on the way of glucose addition (either sterilized in the medium or added aseptically after sterilization). At pH 8.5 and higher the cells begin to form slime and capsules. The strain exhibited growth activity even at initial pH values of the medium 11.5.     

Axenic cultivation of Naegleria gruberi : Requirement for methionine

A simplified axenic medium for Naegleria gruberi strain NEG-M contains -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10⁶ cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH 6.6.     

The electrochemical proton potential of Bacillus alcalophilus.

Bacillus alcalophilus strain ATCC 27647 showed usual growth characteristics, when inoculated at pH 10.4. The cells entered the logarithmic phase at pH 10.3, and as growth continued, the pH dropped further to a value of 8.8 in the stationary phase. B. alcalophilus strain DSM 485 showed comparable growth only in the initial phase after the addition to fresh medium. The small initial growth period was succeeded by a long lag phase, where the pH continuously dropped. The cells resumed growth after the pH was about 10.0 and continued to grow accompanied by a further decrease of external pH. The bioenergetic parameters measured in the initial growth phase of the two strains at high pH (10.1-10.3) were nearly the same, i.e. delta pH = +97 to +110 mV, delta psi = -206 to -213 mV and delta microH+ = -109 to -103 mV. The inverted proton gradient of about 1.7-1.9 at high pH decreased, as the external pH dropped during growth. This led to an increase of the proton motive force (delta microH+), although the membrane potential (delta psi) also declined. The ATP/ADP ratio of strain DSM 485 was high (4.5-5.5) at fast growth during the initial and second growth period. The ratio declined to about 1.5 at the end of the lag phase. At the initial growth phase and at the end of the lag phase, the delta microH+ was, however, the same (approximately -106 mV) and considerably lower than in the middle of the second growth period (approximately -140 mV). Fast growth, therefore, correlates with a high ATP/ADP ratio but not necessarily with a high delta microH+. Addition of gramicidin or carbonylcyanide m-chlorophenylhydrazone stopped growth of B. alcalophilus strain DSM 485 at pH 10.3 or 9.5 and gramicidin immediately decreased the internal ATP/ADP ratio from 4.5 to 1.2 at pH 10.3.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution

Abstract Micrococcus luteus starved for 2–7 months in spent medium following growth to stationary phase in batch culture exhibited a culturability (as estimated by direct plating on nutrient agar plates) of < 0.001%. However, following a lag, some 70% of the cells could be lysed upon inoculation into and cultivation in fresh lactate minimal medium containing penicillin, showing the capability of a significant portion of the cells at least to enlarge (and thus potentially to resuscitate). When the viable cell count was estimated using the most probable number method, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, and little different from the viable cell count as assessed by plating on solid media. However, the apparent viability of these populations evidenced with the most probable number method was 1000–100 000-fold greater when samples were diluted into liquid media containing supernatants taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.