Catabolite repression mutants of Saccharomyces cerevisiae show altered fermentative metabolism as well as cell cycle behavior in glucose-limited chemostat cultures

In glucose-limited continuous cultures, a Crabtree positive yeast such as Saccharomyces cerevisiae displays respiratory metabolism at low dilution rates (D) and respiro-fermentative metabolism at high D. We have studied the onset of ethanol production and cell cycle behavior in glucose-limited chemostat cultures of the wild type S. cerevisiae strain CEN.PK122 (WT) and isogenic mutants, snf1 (cat1) and snf4 (cat3) defective in proteins involved in catabolite derepression and the mutant in glucose repression mig1 (cat4). The triggering of fermentative metabolism was dependent upon catabolite repression properties of yeast and was coincident with a significant decrease of G1 length. WT cells of the strain CEN.PK122 displayed respiratory metabolism up to a D of 0.2 h-1 and exhibited longer G1 lengths than the snf1 and snf4 mutants that started fermenting after a D of 0.1 and 0.15 h-1, respectively. The catabolite derepression mutant snf4 showed a significant decrease in the duration of G1 with respect to the WT. An increase of 300% to 400% in the expression of CDC28 (CDC28-lacZ) with a noticeable shortening in G1 to values lower than approximately 150 min, was detected in the transformed wild type CEN.SC13-9B in glucose-limited chemostat cultures. The expression of CDC28-lacZ was analyzed in the wild type and isogenic mutant strains growing at maximal rate on glucose or in the presence of ethanol or glycerol. Two- to three-fold lower expression of the CDC28-lacZ fusion gene was detected in the snf1 or snf4 disruptants with respect to the WT and mig1 strains in the presence of all carbon sources. This effect was further shown to be growth rate-dependent exhibiting apparently, a threshold effect in the expression of the fusion gene with respect to the length of G1, similar to that shown in chemostat cultures. At the onset of fermentation, the control of the glycolytic flux was highly distributed between the uptake, hexokinase, and phosphofructokinase steps. Particularly interesting was the fact that the snf1 mutant exhibited the lowest fluxes of ethanol production, the highest of respiration and correspondingly, the branch to the tricarboxylic acid cycle was significantly rate-controling of glycolysis.     

Rates of protein synthesis through the cell cycle of Saccharomyces cerevisiae

Exponentially growing cells of Saccharomyces cerevisiae were fractionated by centrifugation in isotonic, self-generated gradients of Percoll. Rapidly growing cells, μ = 0.5 × h⁻¹, with nearly equal length of the daughter and the parental cell cycle were fractionated according to a cell cycle-related density variation. In these cells the net rate of protein synthesis varies nearly 2-fold during the cell cycle. Subsequent separations according to cell size revealed that the highest rate is observed during G2 period. Slow-growing cells, μ = 0.2 × h⁻¹, were fractionated on shallow Percoll gradients in a bimodal fashion, primarily as a dense daughter fraction and a composite light fraction. Thereby a marked high rate of protein synthesis in large unbudded daughter cells was revealed. Separations according to cell size revealed a cell cycle-related separation of budded cells, and the highest rate is observed, as before, in the G2 period. Irrespective of the growth rate a non-exponential increase of cell protein is thereby observed through the cell cycle of budding yeast. Septation and cell separation coincide with a low degree of ribosome exploitation.     

Enhanced lipid production in Nannochloropsis sp. using fluorescence‐activated cell sorting

To advance the utilization of microalgae as a viable feedstock for biodiesel production, the intracellular lipid content of three strains of the marine microalgae Nannochloropsis sp. was enhanced using flow cytometry (FC) coupled with cell sorting. Total lipid content was doubled to 55% (biomass dry weight) in the sorted, daughter cells of Nannochloropsis (strain 47) after consecutive three rounds of cell sorting, and this trait was maintained for approximately 100 subsequent cell generations. In addition, daughter cells had a fatty acid profile similar to that of the parent, wild‐type strain. The study demonstrates that FC coupled with cell sorting is a powerful tool for the enhancement of intracellular lipid content in microalgae exploited for biodiesel feedstock.     

Non‐equivalence in old‐ and new‐flagellum daughter cells of a proliferative division in Trypanosoma brucei

Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new‐flagellum and the old‐flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new‐flagellum daughter in particular re‐modelling rapidly and extensively in early G1. This re‐modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old‐flagellum daughter undergoes a different G1 re‐modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non‐equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non‐equivalence.     

The impact of MIG1 and/or MIG2 disruption on aerobic metabolism of succinate dehydrogenase negative Saccharomyces cerevisiae

The zinc finger proteins Mig1 and Mig2 play important roles in glucose repression of Saccharomyces cerevisiae. To investigate whether the alleviation of glucose effect would result in an increase in aerobic succinate production, MIG1 and/or MIG2 were disrupted in a succinate dehydrogenase (SDH)-negative S. cerevisiae strain. Moreover, their impacts on physiology of the SDH-negative S. cerevisiae strain were studied under fully aerobic conditions when glucose was the sole carbon source. Our results showed that the succinate production for the SDH-negative S. cerevisiae was very low even under fully aerobic conditions. Furthermore, deletion of MIG1 and/or MIG2 did not result in an increase in succinate production in the SDH-negative S. cerevisiae strain. However, the synthesis of acetate was significantly affected by MIG1 deletion or in combination with MIG2 deletion. The acetate production for the mig1/mig2 double mutant BS2M was reduced by 69.72% compared to the parent strain B2S. In addition, the amount of ethanol produced by BS2M was slightly decreased. With the mig2 mutant BSM2, the concentrations of pyruvate and glycerol were increased by 26.23% and 15.28%, respectively, compared to the parent strain B2S.     

Expression of the SNF1 gene from Candida tropicalis is required for growth on various carbon sources, including glucose

SNF1 of Saccharomyces cerevisiae is an essential gene for the derepression of glucose repression. A homolog of SNF1 (CtSNF1) was isolated from an n-alkane-assimilating diploid yeast, Candida tropicalis. CtSNF1 could complement the snf1 mutant of S. cerevisiae. The previously published method for introducing the exogenous DNA into C. tropicalis was employed to construct SNF1/ snf1 heterozygote and snf1/snf1 homozygote strains. The successfully constructed SNF1/snf1 heterozygote was named KO-1. Disruption of the second CtSNF1 allele was unsuccessful, suggesting that CtSNF1 might be essential for cell viability. Therefore, in order to control the expression of CtSNF1, a strain (named KO-1G) in which the promoter region of CtSNF1 was replaced with the GAL10 promoter of C. tropicalis was constructed, and the growth of strains KO-1 and KO-1G was compared with that of the parental strain. The growth of strain KO-1 on glucose, sucrose, or acetate did not differ from the growth of the parental strain, but strain KO-1 showed a slight growth retardation on n-alkane. The growth of strain KO-1G on galactose was normal, but the cells stopped growing when transferred to glucose-, acetate-, or n-alkane-containing medium. Northern blot analysis against mRNA from the n-alkane-grown KO-1G strain demonstrated a close relationship between the presence of CtSNF1 mRNA and the growth of the cells, indicating that CtSNF1 is essential for cell viability. Moreover, mRNA levels of isocitrate lyase, which is localized in peroxisomes of C. tropicalis, were significantly affected by the level of CtSNF1 mRNA. Received: 3 May 1999 / Accepted: 14 July 1999     

Age-related changes in the physiological state of budding yeast cells

Biochemical and physiological changes occurring during the relative ageing of budding yeast cells were studied. In contrast to scar-free daughter cells the scar-bearing mother cells display a longer G1 phase of the cell cycle, synthesize RNA more slowly, consume oxygen more rapidly, possess a higher saturation constant K_s, and their cell walls are less resistant to mechanical strain.     

Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division

The budding yeast, Saccharomyces cerevisiae, was grown exponentially at different rates in the presence of growth rate-limiting concentrations of a protein synthesis inhibitor, cycloheximide. The volumes of the parent cell and the bud were determined as were the intervals of the cell cycle devoted to the unbudded and budded periods. We found that S. cerevisiae cells divide unequally. The daughter cell (the cell produced at division by the bud of the previous cycle) is smaller and has a longer subsequent cell cycle than the parent cell which produced it. During the budded period most of the volume increase occurs in the bud and very little in the parent cell, while during the unbudded period both the daughter and the parent cell increase significantly in volume. The length of the budded interval of the cell cycle varies little as a function of population doubling time; the unbudded interval of the parent cell varies moderately; and the unbudded interval for the daughter cell varies greatly (in the latter case an increase of 100 min in population doubling time results in an increase of 124 min in the daughter cell's unbudded interval). All of the increase in the unbudded period occurs in that interval of G1 that precedes the point of cell cycle arrest by the S. cerevisiae alpha-mating factor. These results are qualitatively consistent with and support the model for the coordination of growth and division (Johnston, G. C., J. R. Pringle, and L. H. Hartwell. 1977. Exp. Cell. Res. 105:79-98.) This model states that growth and not the events of the DNA division cycle are rate limiting for cellular proliferation and that the attainment of a critical cell size is a necessary prerequisite for the "start" event in the DNA-division cycle, the event that requires the cdc 28 gene product, is inhibited by mating factor and results in duplication of the spindle pole body.     

A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

ABSTRACT By adding the protein synthesis inhibitor, emetine (10^-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.     

GROWTH CHARACTERISTICS OF MARINE PHYTOPLANKTON DETERMINED BY CELL CYCLE PROTEINS: THE CELL CYCLE OF ETHMODISCUS REX (BACILLARIOPHYCEAE) IN THE SOUTHWESTERN NORTH ATLANTIC OCEAN AND CARIBBEAN SEA1

Ethmodiscus spp. is an important contributor to oceanic tropical-ooze sediments and thus might be an important transport vehicle of carbon from the ocean surface to sediments. The knowledge of its cell cycle and growth rate, which is still lacking, is necessary to evaluate the importance of Ethmodiscus in nutrient cycling and to solve the discrepancy between its high sedimentary abundance and rarity in the plankton. We used immunofluorescence of a cell cycle protein, prolqerating cell nuclear antigen (PCNA), and DNA-specific staining to study the progression of the cell cycle and roughly estimate the growth rate for E. rex (Rattray) Wiseman and Hendey in the southwestern North Atlantic Ocean and Caribbean Sea in June 1994 and January 1995. During the cell division cycle, the chloroplasts appeared to synthesize DNA before the nucleus (S phase). Following the S phase, the nucleus moved from one end of the cell toward the center underneath the midline of the girdle band (G2 phase) where it divided (M phase). During a very brief period, the parent cell split and moved apart from the girdle midline, and two new valves were produced (late M phase). The two daughter nuclei apparently remained attached at the joint of the two newly produced valves, where they appeared to be responsible for coordinating the symmetrical formation of the new valves. The morphologically complete daughter cells remained joined for a short period of time before separating into solitary cells whose nucleus was located at one end of the cell. Derived from the phase fraction curves, the duration of the cell cycle phases decreased in the order from G1, S, G2, to M. A conservative estimate of the growth rate in the study area obtained by using PCNA immunostaining was 0.39–0.46 d^?1 in June and 0.15 d^?1 in January. The validity and implication of the growth rate estimates are discussed.     

Flow Cytometry studies of marine phytoplankton populations

Abstract

Flow cytometry has been applied to the study of phytoplankton populations and cell components. In this work, cells cycle studies on three marine diatoms cultures were carried out by a flow cytometer. Phaeodactylum tricomutum, Chaetoceros simplex and Thalassiosira allenii, showed different DNA patterns in G1, G2, S, phases. This situation may be related to the specific algal physiology. Cultures of P. tricomutum were maintained in 4 media with different silicates concentrations. The lack of silicates did not seem produced a significative cells arresting in the cell cycle phases. During the experiment, the cell fraction in S phase, decreased in all media tested. These preliminary results could be further developed to apply flow cytometry to environmental problems in order to identify general algal groups and study algal physiology in different growth conditions.     

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways^1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time^3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein⁶. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured^7-9.The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.     

EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.     

Characterization of 14 different putative protein kinase cDNA clones of the C4 plantSorghum bicolor

C₄ photosynthesis is functionally dependent on metabolic interactions between mesophyll- and bundle-sheath cells. Although the C₄ cycle is biochemically well understood, many aspects of the regulation of enzyme activities, gene expression and cell differentiation are elusive. Protein kinases are likely involved in these regulatory processes, providing links to hormonal, metabolic and developmental signal-transduction pathways. Here we describe the cloning and characterization of 14 different putative protein kinase leaf cDNA clones from the C₄ plant Sorghum bicolor. These genes belong to three different protein kinase subfamilies: ribosomal protein S6 kinases, SNF1-like protein kinases, and receptor-like protein kinases. We report the partial cDNA sequences, mesophyll/bundle-sheath steady-state mRNA ratios, mesophyll/etiolated leaf steady-state mRNA ratios, and the positions of 14 protein kinase genes on the genetic map of S. bicolor. Only three of the protein kinase genes described here are expressed preferentially in mesophyll cells as compared with the bundle-sheath. Received: 16 January 1998 / Accepted: 3 April 1998     

Daughter cells as an important factor in determining the physiological state of yeast populations

Cells of Candida utilis grown in a single-stage chemostat at D = 0.05, 0.1, 0.25, and 0.35 hr^?l were separated into a fraction of scar-bearing mother cells and a fraction of scar-free daughter cells. The scar-free cells were transferred into small batch cultures where the length of the maturation phase, changes in length and width of cells, specific growth rate, and specific rate of RNA and protein synthesis were examined for 5 hr. The daughter cells grown at D = 0.05 hr^?1 were very small at the moment of separation from the mother cells (about one-third of the mother cell). Their maturation phase (in a batch culture), at the beginning of which they attain the specific growth rate approaching the μ_max of the strain used, lasts for 3 hr. On the other hand, daughter cells grown at D = 0.35 hr^?1 are almost the same size as the mother cells at the moment of separation. After transfer to a batch culture they begin to bud almost immediately. Similarly, in their other morphological and physiological parameters they differ strikingly from immature daughter cells which are formed at low specific growth rates. The importance of these differences from the point of view of mathematical modeling of growth processes is discussed.