ROLE OF THE LIGHT-DARK CYCLE AND MEDIUM COMPOSITION ON THE PRODUCTION OF COCCOLITHS BY EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Production of coccoliths by cells of Emiliania huxleyi (Lohmann) Hay and Mohler was measured during exposure of the cells to two diel light-dark cycles (16:8 h). During the light period about eight coccoliths per cell were formed at a constant rate of one coccolith per 2 h. Cells divided during the first half of the dark period. No coccolith production took place during the dark period. With electron microscopy we found early-stage, coccolith-production compartments in cells after mitosis while still in the dark. No calcification was observed in these compartments. Cells grown on enriched seawater (Eppley's medium) tended to produce enough coccoliths to cover the cell in a single layer. When these cells reached the stationary phase coccolith production stopped. Coccolith production was induced by removal of extracellular coccoliths. Cells grown on medium containing 2% of the nitrate and phosphate of Eppley's medium tended to produce coccoliths in the stationary phase. This resulted in the formation of multiple layers of coccoliths. The multiple covering was restored after decalcification of stationary cells. Formation of multiple layers of coccoliths may help the cells reach deeper, nutrient-rich water by increasing the sinking rate of the cells.     

Das Adenosinphosphat-System während Wachstum und Entwicklung von Acanthamoeba castellanii

The concentration of adenosine tri-, adenosine di-, and adenosine monophosphate in cells of Acanthamoeba castellanii was measured during the logarithmic growth phase and the stationary growth phase in which trophozoites were transformed into cysts. This developmental process was induced in three ways: by growth in nutrient medium to high cell density, by transferring cells in the logarithmic phase into a nutrient-free medium, and by mixing logarithmically growing cells with ethidium bromide. In all cases, encystment is accompanied with a reduction of total adenosine phosphate content to about 85%, mainly because of a depletion of cellular ATP. The value of the adenosine phosphate energy charge in logarithmically growing amoebae is 0.83. During development the energy charge becomes stabilized at different values (between 0.58 and 0.81), characteristic to the mode of encystation. A possible functional relationship between changes of the adenosine phosphate concentration and developmental processes of the amoeba is discussed.     

Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.     

Cohesive properties of axenically grown cells of the slime mould Dictyostelium discoideum

It has been found that Dictyostelium discoideum cells from the exponential growth phase of axenically grown cultures are cohesive, whereas those from stationary phase are not. These differences in cohesiveness are seen in phosphate buffer and in axenic medium. Stationary phase medium inhibits the aggregation of log phase cells; stationary phase cells inoculated into freshly prepared medium regain their cohesiveness. Stationary phase medium may contain an inhibitor of cell cohesion. pH differences between the two types of medium are not entirely responsible for loss of cohesiveness.     

Renouvellement des protéines et effet spécifique de la kinétine sur des cultures de cellules de Tabac

Determination and fractionation of proteins of tobacco cell suspensions requireing kinetin for cell division. — Cell suspensions either in stationary phase without kinetin in the medium or dividing in the presence of this factor have been compared. It was found a) that the specific rates of protein synthesis and protein degradation were not changed by the addition of kinetin during the early period of growth. A quantitative change ocurred only after the first generation period, b) Soluble proteins of these cells were mapped by polyacrylamide gel electrophoresis. The observed protein patterns were very similar as well as the patterns or radioactivity incorporated into the same proteins during the incubation period of the cells. However, a small number of specific discrepancies appeared in the pattern of cells growing in the presence of kinetin matched to the patterns of stationary cells. At least one specific difference in these patterns could be observed before the first cell division occurred.     

Nutrient uptake and accumulation by sugarcane cell cultures in relation to the growth cycle

Growth characteristics and nutrient changes in medium and cells of batch-grown sugarcane cultures were investigated over a period of 14 days. Amino acids, PO ₄ ³⁻ and K⁺ were substantially removed from the medium during the first seven days of culture; a strong preference for uptake of organic nitrogen over inorganic nitrogen was observed. Sodium uptake increased during the time when K⁺ was becoming deficient in the medium. The main anions taken up were SO ₄ ²⁻ and PO ₄ ³⁻ . Strong acidification and a virtually total extracellular hydrolysis of sucrose in the medium during the first seven days of culture were also observed. Tapering off of the rapid growth phase was accompanied by an increase of intra-cellular sucrose and a decrease of intracellular protein. As cells went from rapid growth into stationary phase, cytoplasmic space of the cells decreased slightly in favor of vacuolar space. Overall cell volume stayed constant throughout the growth cycle, except during a short period before onset of rapid growth. Transport of the glucose analog 3-O-methyl glucose remained constant in terms of K_m value but the V_max was slightly higher in rapidly growing cells. Published with the approval of the Director as paper no. 495 in theJournal Series of the Experiment Station, Hawaiian Sugar Planters' Association     

Recovery from nutrient starvation by a marine Vibrio sp

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.     

Recovery from nutrient starvation by a marine Vibrio sp.

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Biomass relationship to growth and phosphate uptake ofPseudomonas fluorescens,Escherichia coli andAcinetobacter radioresistens in mixed liquor medium

The ability ofPseudomonas fluorescens, Escherichia coli andAcinetobacter radioresistenns to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10²–10⁵ cells ml^–1) were used. At a high initial biomass concentration (10⁸ cells ml^–1), phosphate was removed during the lag phase and during logarthmic growth byP. fluorescens. Escherichia coli. at high initial biomass concentrations (10⁷ cells ml^–1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantily of phosphate during the stationary growth phase.Acinetobacter radioresistens, at high initial cell densities (10⁶, 10⁷ cells ml^–1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase.Pseudomonas fluorescens removed phosphate more thanA. radioresistens andE. coli with specific average ranges from 3.00–28.50 mg L^–1 compared to average ranges of 4.92–17.14 mg L^–1 forA. radioresistens and to average ranges of 0.50–8.50 mg L^–1 forE. coli.     

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.     

Wachstum und N-Stoffwechsel sowie Anderungen des Isoenzymspektrums der Glutamatdehydrogenase (GDH) in batchvermehrten, licht- und dunkelkultivierten Zellsuspensionen von Pisum sativum

Growth, N-metabolism and isoenzyme pattern of glutamate dehydrogenase in batch-cultures of Pisum sativum cells under light and dark conditions. Cell suspension cultures of Pisum sativum L. derived from root and shoot sections of seedlings have been prepared and cultured in defined nutrient medium. Both the cells and the media were analysed daily for the N-fractions and carbohydrates during the growth period. The data obtained indicate specific correlations between growth and nitrogen and carbohydrate metabolism. At the beginning of the growth cycle ammonia as compared to nitrate was favoured in uptake. An increased uptake of nitrate occurred at the end of the linear growth phase when carbohydrate in the media was depleted. The uptake of sucrose was rapid during the whole growth cycle, only in the range of the linear growth phase the uptake stagnated for 3 or 4 days. During increased biosynthesis of nitrogenous compounds at the beginning of the growth cycle up to seven isoenzymes of the glutamate dehydrogenase could be separated by polyacrylamide gel electrophoresis. The isoenzyme pattern changed during the stationary growth phase, especially when the carbohydrate content in the medium decreased. There is some evidence that the isoenzyme pattern is influenced by carbohydrate metabolism.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

HSP12, a new small heat shock gene of Saccharomyces cerevisiae: Analysis of structure, regulation and function

Summary We have isolated a new small heat shock gene, HSP12, from Saccharomyces cerevisiae. It encodes a polypeptide of predicted Mr 12 kDa, with structural similarity to other small heat shock proteins. HSP12 gene expression is induced several hundred-fold by heat shock and on entry into stationary phase. HSP12 mRNA is undetectable during exponential growth in rich medium, but low levels are present when cells are grown in minimal medium. Analysis of HSP12 expression in mutants affected in cAMP-dependent protein phosphorylation suggests that the gene is regulated by cAMP as well as heat shock. A disruption of the HSP12 coding region results in the loss of an abundant 14.4 kDa protein present in heat shocked and stationary phase cells. It also leads to the induction of the heat shock response under conditions normally associated with low-level HSP12 expression. The HSP12 disruption has no observable effect on growth at various temperatures, nor on the ability to acquire thermotolerance.     

The intracellular Na+ and K+ composition of the moderately halophilic bacterium, Paracoccus halodenitrificans

The intracellular concentrations of Na+ and K+ in exponentially growing Paracoccus halodenitrificans were independent of the NaCl concentration of the growth medium. The observed values were approximately 100 and 300 mM for Na+ and K+, respectively. In stationary phase cells, the ultimate values for Na+ depended on the NaCl concentration of the growth medium. With cells grown in the presence of 1 M NaCl, the value was about 500 mM; for cells grown in the presence of 3 M NaCl, the value was about 1.1 M. The K+ concentration in stationary phase cells was unaffected by the NaCl concentration in the growth medium. The final value was about 100 mM. Associated with these changes were changes in the ATP pool and decreases in the activities of the NADH oxidase system and the membrane-bound ATPase. It is proposed that the decrease in the activities of these enzymes may account for the ion flows observed in stationary phase cells.     

Factors influencing the activity of cellular alkaline phosphatase during growth and sporulation ofBacillus cereus

Alkaline phosphatase (EC 3.1.3.1) is synthesized in media with a low phosphate concentration (0.37 mmm of total and 19 μm of inorganic phosphate, respectively) already during the exponential phase of growth ofBacillus cereus. The enzyme is repressed by higher phosphate concentrations (3.7 mm) during the whole growth period; during sporogenesis the enzyme activity in cells slightly increases even under these conditions. During growth the enzyme is not secreted into the medium, a minor amount being released after cessation of growth. The enzyme activity can be increased by adding Zn²⁺ ions (10 μm). When during growth without phosphate the pH of the medium decreases below 5.0, the enzyme activity temporarily decreases and growth is slowed down, followed by a subsequent increase of the enzyme activity. In this case the onset of sporulation is also delayed.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Disappearance of Mating Reactivity in Paramecium caudatum upon Repeated Washing

SYNOPSIS The appearance of mating reactivity of Paramecium caudatum was retarded by repeated washing of the cells in the logarithmic growth phase with Dryl's and other solutions. Highly reactive cells in the stationary phase also lost reactivity during the same treatment. Dryl's solution, sodium and potassium phosphate buffers, Miyake's physiologic balanced solution and exhausted culture medium were effective but deionized water saturated with CaCO₃ was ineffective. The addition of supernatant fluid from stationary phase cultures restored reactivity in 2 hr but was unable to do so when applied to extremely starved cells. These findings may be useful for study of the synthesis of mating-type substances.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN IN LIGHT/DARK CYCLES. II. PHASED PO4-LIMITED CULTURES1

The characteristics of phosphate uptake and photosynthetic capacity were studied in P-limited populations of Euglena gracilis Klebs (Z), using both P-limited batch cultures in stationary phase and cyclostat cultures grown on 14:10 LD. P uptake obeyed Michaelis-Menten kinetics between 0 and 150 μM PO₄ under both growth conditions. The value of V_max was 35% lower in the dark than in the light in the stationary phase cells. The value of K₈ was not affected by light conditions, and uptake was completely inhibited in the presence of 1 mm KCN. P uptake (at 2.0 μM PO₄) and photosynthetic capacity showed diel periodicity with peak rates occurring just before the beginning of the dark period for P uptake, and 8 h into the light period for photosynthetic capacity. V_max for P uptake increased by a factor of 1.5 over the light period, whereas K₈ remained constant at 1.4 μM PO₄. These patterns were displayed by both nondividing stationary phase cells and populations in which less than a third of the cells divided each day, indicating that the rhythmicity is not coupled to cell division.