Ion metabolism in a Halobacterium. I. Influence of age of culture on intracellular concentrations

Work is described on the changes in cell ions during growth of cultures of a species of Halobacterium isolated from the Dead Sea. Cell K concentration fell from 5.5 to 3.8 moles per kg cell water during the logarithmic phase of growth and maintained the latter value during the stationary phase (initial medium concentration, 7 mM). Cell Na and Cl followed a complex series of roughly parallel changes. The logarithmic phase ion concentrations were: Na, 1.0–2.3 moles/kg cell water; Cl, 2.3–3.7 moles/kg cell water. The final stationary phase values were: Na, 0.5 moles/kg cell water; Cl, 2.3–2.9 moles/kg cell water (medium NaCl concentration, 3.9 Molal). It is suggested that most of the K⁺ is bound within the cytoplasm.     

Cation Transport in Escherichia coli : I. Intracellular Na and K concentrations and net cation movement

Methods have been developed to study the intracellular Na and K concentrations in E. coli, strain K-12. These intracellular cation concentrations have been shown to be functions of the extracellular cation concentrations and the age of the bacterial culture. During the early logarithmic phase of growth, the intracellular K concentration greatly exceeds that of the external medium, whereas the intracellular Na concentration is lower than that of the growth medium. As the age of the culture increases, the intracellular K concentration falls and the intracellular Na concentration rises, changes which are related to the fall in the pH of the medium and to the accumulation of the products of bacterial metabolism. When stationary phase cells, which are rich in Na and poor in K, are resuspended in fresh growth medium, there is a rapid reaccumulation of K and extrusion of Na. These processes represent oppositely directed net ion movements against concentration gradients, and have been shown to be dependent upon the presence of an intact metabolic energy supply.     

The Mechanism of Onset of the Stationary Phase in Euglena gracilis Grown with 10 mM Succinate: Intracellular pH Values*

SYNOPSIS. When Euglena gracilis were grown with 10mM succinate at pH 3.5 the extracellular pH averaged 3.62 and the cultures had produced 6 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 30 μliters/10⁶ cells/hr. Total protein and dry weights per cell remained constant during the logarithmic phase and began to decline when the late logarithmic phase was reached. Added succinate caused the cultures in stationary phase to commence logarithmic growth once more. Onset of the stationary phase in cultures grown at pH 3.5 was due to depletion of succinate. When cultures were grown at pH 6.9 the extracellular pH averaged 7.62 and the cultures produced 3 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 20 μliters/10⁶ cells/hr during the logarithmic phase. The decline in total protein and dry weights per cell began at the beginning of the logarithmic phase and continued into the stationary phase of growth. Cultures grown at pH 3.5 should produce a larger number of cells/ml than cultures grown at pH 6.9 if the cells are responding to the unionized moiety of succinate and not the ionized moiety. At pH 3.5 83% of the succinate is unionized, whereas at pH 6.9 0.20% of the succinate is unionized. The onset of the stationary phase in cultures grown at pH 3.5 and pH 6.9 is due to lack of an adequate amount of extracellular unionized succinate. Intracellular pH values were determined in cultures grown at pH 6.9 using the weak acid DMO (5.5-dimethyl-2,4-oxazolidinedione). As the extracellular pH increased from 6.90 to 7.62, the intracellular pH increased from 5.89 to 6.89. As the extracellular pH increased from 7.62 to 8.44, the intracellular pH increased from 6.89 to 7.50.     

Electrophysiology of an Insect Heart

Bioelectric activity in single cells of the moth myocardium has been measured in situ with intracellular microelectrodes with particular reference to the bizarre ionic medium which bathes the tissues. Resting potentials averaged 47 mv, inside negative with respect to outside, despite a value of 11 mv calculated on the basis of transmembrane potassium concentration gradients. Action potentials overshoot as much as 12 mv in the virtual absence of extracellular sodium. Two "types" of action potentials have been recorded; one that resembles vertebrate atrial action potentials is found in the cephalic region of the tubular heart, and the other, similar in contour to vertebrate ventricular action potentials, is found in the areas posterior to the first abdominal segment. Histological sections indicate no structural differences between the two areas. Typical cardiac pacemaker type potentials occur but are not topographically localized. The effects of the omission from the perfusion fluid of the four major cationic constituents, Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺, on resting and action potentials may be summarized as: no effect, hyperpolarization, prolonged repolarization, and depolarization, respectively.     

Electrical Properties of Neurospora crassa : Effects of external cations on the intracellular potential

Glass micropipette electrodes have been employed to study the transsurface potential difference of Neurospora crassa. For mature hyphae grown in agar cultures, the internal potential is large and negative, often exceeding -200 mv. The potential is sensitive to the concentrations of extracellular potassium, sodium, hydrogen, and calcium ions, but does not vary in a manner which is readily explained by ionic diffusion potentials. With extracellular solutions containing only potassium chloride (or sulfate) and sucrose, the internal potential shifts toward zero (becomes less negative) at 45 mv per tenfold increase of potassium, over the range 0.1 to 10 mM. A similar result has been found with sodium, though the slope is only 33 mv/log unit. Calcium (1 mM) diminishes the influence of potassium and sodium by 60 to 70 per cent. As potassium or sodium is raised above 20 mM, the slope of the internal potential increases sharply to 85 to 90 mv/log unit, both in the presence and absence of calcium. With increasing hydrogen ion concentration, too, the internal potential shifts toward zero; in this case the slope is about 12 mv/pH unit at pH 9 and rises smoothly to 33 mv/pH unit at pH 3. All these phenomena are probably properties of the plasma membrane. The polysaccharide cell wall contains few fixed negative charges, has a low transverse resistance, and supports very little potential difference when separated from the plasma membrane.     

Cation Transport in Escherichia coli : IV. Kinetics of net K uptake

The resuspension of K-poor, Na-rich stationary phase E. coli in fresh medium at pH 7.0 results in a rapid uptake of K and extrusion of Na by the cells. In all experiments net K uptake exceeded net Na extrusion. An investigation of the uptake of glucose, PO₄, and Mg and the secretion of H by these cells indicates that the excess K uptake is not balanced by the simultaneous uptake of anions but must be accompanied by the extrusion of cations from the cell. The kinetics of net K uptake are consistent with the existence of two parallel influx processes. The first is rapid, of brief duration, and accounts for approximately 60 per cent of the total net K uptake. This process is a function of the extracellular K concentration, is inhibited in acid media, and appears to be a 1 for 1 exchange of extracellular K for intracellular H. The second influx process has a half-time of approximately 12 minutes, and is not affected by acid media. This process is a function of the intracellular Na concentration, is dependent upon the presence of K in the medium, and may be ascribed to a 1 for 1 exchange of extracellular K for intracellular Na.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.     

Surface electrokinetic properties of two strains of Staphylococcus aureus during their cell growth cycle

Two strains of Staphylococcus aureus were investigated: S. aureus H, a normal wild-type strain, and 52A5, a mutant strain whose cell wall contains no teichoic acid but is made up entirely of mucopeptide. S. aureus H cells in the lag or stationary phase of growth had an electrophoretic mobility of ?1.10 μm/s/V/cm while those in the logarithmic phase had a mobility of ?0.80 μm/s/V/cm in saline at pH 7.2, 0.6 mM NaHCO₃, 25°C (I = 0.145 g-ions/l). S. aureus 52A5 cells in the same solution had a mobility of ?0.87 μm/s/V/cm in lag and stationary growth phases but a mobility of ?1.30 μm/s/V/cm in the logarithmic growth phase. The S. aureus H cell surfaces at lag phase had pKs of 3.2 and 9.5; at logarithmic phase, 4.2 and 9.0; and at stationary phase, 3.0 and 9.5. The 52A5 cell surfaces at lag phase had pKs of 2.3 and 10.3; at logarithmic phase, 1.7 and 8.5; at stationary phase, 2.6 and 10.2.     

The production of extracellular carbohydrates by estuarine benthic diatoms: the effects of growth phase and light and dark treatment

Epipelic diatoms are important constituents of estuarine microphytobenthic biofilms. Field‐based investigations have shown that the production of carbohydrates by such taxa is ecologically important. However, limited information exists on the dynamics of carbohydrate production by individual species of epipelic diatoms. The production of low and high molecular weight extracellular carbohydrates in axenic cultures of five species of benthic estuarine diatoms, Cylindrotheca closterium (Ehrenberg), Navicula perminuta (Grun.) in Van Heurck, Nitzschia frustulum (Kütz.) Grunow, Nitzschia sigma (Kütz.) Grunow, and Surirella ovata (Kütz.) Grunow, were investigated. All species produced colloidal (water‐soluble) carbohydrates during growth, with maximal production occurring during stationary phase. During logarithmic growth, approximately 20% of extracellular carbohydrates consisted of polymeric material (extracellular polymeric substances [EPS]), but during stationary phase, EPS content increased to 34%–50%. Pyrolysis–mass spectrophotometry analysis showed differences in the composition of EPS produced during logarithmic and stationary phase. All species synthesized glucan as a storage carbohydrate, with maximum glucan accumulation during the transition from log to stationary phase. Short‐term labeling with ¹⁴C‐bicarbonate found that between 30 and 60% of photoassimilates were released as colloidal carbohydrate, with EPS consisting of approximately 16% of this colloidal fraction. When cells were placed in darkness, EPS production increased, and between 85 and 99% of extracellular carbohydrate produced was polymeric. Glucan reserves were utilized in dark conditions, with significant negative correlations between EPS and glucan for N. perminuta and S. ovata. Under dark conditions, cells continued to produce EPS for up to 3 days, although release of low molecular weight carbohydrates rapidly ceased when cells were dark treated. Three aspects of EPS production have been identified during this investigation: (1) production during rapid growth, which differs in composition from (2) EPS directly produced as a result of photosynthetic overflow during growth limiting conditions and (3) EPS produced for up to 3 days in the dark using intracellular storage reserves (glucans). The ecological implications of these patterns of production and utilization are discussed.     

Adenylate cyclase activity of Bordetella organisms. I. Its production in liquid medium

Abundant adenylate cyclase activity was found in the phase I cultures not only of Bordetella pertussis but also fo B. parapertussis and B. bronchiseptica. The enzyme activity in the culture fluid increased rapidly and reached a peak during the logarithmic growth phase. B. parapertussis and B. bronchiseptica especially produced a high activity of the enzyme in the culture fluid during the logarithmic phase, but little or no activity was detected in the cells throughout the growth period. In the culture of B. pertussis, the intracellular activity was higher than that in the culture fluid. Phase III cultures of these species lacked both the extracellular and intracellular enzyme activities throughout their growth. In the culture of B. parapertussis, accumulation of cyclic AMP was parallel to that of adenylate cyclase activity through the growth periods, but in B. pertussis there was no parallelism from the stationary through the declining phases. The difference in production patterns of the enzyme activity among the species is discussed.     

Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture

Three species of microalgae were grown in mass culture to investigate the influence of culture technique and growth phase on the production of 20:5(n?3) and 22:6(n?3). These polyunsaturated fatty acids (PUFA) are considered to be essential in many marine animals diets for high growth and survival rates. The species of microalgae examined wereNannochloropsis oculata, Pavlova lutheri andIsochrysis sp. (clone T.Iso). All batch cultures (logarithmic and stationary phase) and semi-continuous cultures (logarithmic phase) examined contained high levels of the long-chain (n?3) PUFA, but production could be maximised by harvesting at specific times and growth phases. Maximum cellular content (pg cell^-1) of long-chain PUFA was found in logarithmic phase batch cultures ofN. oculata and in stationary phase cultures ofP. lutheri. The cellular content of PUFA in cultures ofIsochrysis sp. did not change significantly with culture technique or growth phase. Alternatively, stationary phase cultures of all three species showed increased proportions (%) and cellular contents of triacylglycerols, and saturated and monounsaturated fatty acids with correspondingly decreased proportions of polar lipids and most PUFA relative to logarithmic phase cultures. The exception was the proportion and cellular content of 22:6(n?3) inP. lutheri which increased with triacylglycerol content. The mass of long-chain (n?3) PUFA per volume of culture was significantly higher in stationary phase cultures due to the higher cell counts per volume. These findings indicate that the opportunity exists to maximise PUFA production by microalgae with the potential to improve animal growth and reduce production costs in mariculture operations and may be of use in the large scale culture and harvesting of microalgae for the biotechnology industry.     

Excretion of a protease by Serratia marcescens

Excretion of an extracellular protease of Serratia marcescens ATCC 25419 occurred during logarithmic growth and was highest (per cell) when cultures reached the stationary growth phase. Production of the extracellular protease was induced by leucine or casein in minimal medium or by growth in tryptone-yeast medium. In the late stationary phase an intracellular protease activity accumulated which was also observed in mutants with very low extracellular protease activity. The excreted protease was the dominant protein in the growth medium. The protease was purified to homogeneity by column chromatography on Bio-Gel P-100 and on DEAE-cellulose. Quantitative amino acid analysis revealed the absence of sulfurcontaining amino acids. The enzyme consists of one polypeptide chain. A molecular weight of 51,000 and 55,000 was estimated using polyacrylamide gel electrophoresis and chromatography on Bio-Gel P-100 respectively. The enzyme cleaved only N--benzoyl-DL-lysine-and-arginine-nitroanilides but not the corresponding leucine or tyrosine derivatives nor a set of diand tripeptides.Abbreviation SDS sodium dodecylsulfate     

Glucose transport in nongrowing yeast

The inducible, nonenergy-requiring glucose transport system of the yeast Kluyveromyces lactis is inactivated upon starving cells of glucose by (1) transferring logarithmic phase glucose-grown cells to synthetic medium containing a nonglycolytic carbon source, and (2) upon transition of logarithmic phase glucose-grown cells to stationary phase. The steady-state accumulation of nonmetabolizeable 6-deoxyglucose and the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of transport of 6-deoxyglucose is the same in stationary phase cells and in logarithmic phase cells. The rate of transport is lower in the nongrowing cells. Restoration of activity requires energy and protein synthesis as well as inducer.     

Production of intracellular enzymes by enzymatic treatment of yeast

Enzymatic extraction of intracellular enzymes from various yeasts by glucanase was investigated. Favourable conditions for lysis and release of intracellular enzymes were established. The effects of yeast concentration, growth phase of yeast, storage temperature and pretreatment of yeast were studied. The yeasts investigated can be divided into two groups. The first, Kluyveromyces lactis, Saccharomyces cerevisiae, Saccharomyces oviformis, Torulopsis glabrata, Hansenula polymorpha and local bakers' yeast, lysed relatively easily (70–80% of the cells), especially when cells from the logarithmic growth phase were treated. The second, Candida utilis and Candida vini, were more susceptible to lysis (40–50%) when cells were taken from the stationary phase. Release of two enzymes, glycerol kinase from Candida utilis grown on glycerol and formate dehydrogenase from Torulopsis glabrata grown on methanol was examined. The highest specific activities were obtained by incubating the cells with glucanase for 1.5 h at 37°C. Inactivation of the released enzyme was relatively low. After 12 h of enzymatic treatment at 28°C glycerol kinase maintained about 50%, and formate dehydrogenase over 80%, of the original activities.     

Characteristics of intracellular metabolism of procollagen and other proteins in human embryo fibroblasts in trisomy for chromosome 7

A comparative study of the relative rates of intracellular total protein metabolism in diploid and aneuploid (with trisomy for chromosome 7) human embryo fibroblasts in the logarithmic and stationary growth phases was carried out. Using double labeling with [14C]proline (24 hrs) and [3H]proline (3 hrs), it was found that: the rates of intracellular protein metabolism during transition to the stationary phase of growth are increased in diploid cells and decreased in cells with trisomy for chromosome 7; the relative rate of protein metabolism in the logarithmic phase is higher in trisomic cells than in diploid ones. The intracellular degradation of procollagen in trisomic cells is increased approximately by 17% as compared to normal fibroblasts. Treatment of cell lysates with bacterial collagenase revealed the presence of procollagen incomplete degradation products in anomalous fibroblasts. The observed differences in the rates and mode of protein metabolism during transition of diploid and trisomic fibroblasts to the stationary phase of growth suggest that the odd autosome interferes with the normal coordinated activity of genes in chromosomes.     

Characterization of nucleotide pools as a function of physiological state in Escherichia coli

Using a modified method that involves minimal manipulation of cells, we report new information about nucleotide pool sizes and changes throughout the Escherichia coli growth curve. Nucleotide pool sizes are critically dependent on sample manipulation and extraction methods. Centrifugation and even short (2 min) lapses in sample preparation can dramatically affect results. The measured ATP concentration at three different growth rates is at least 3 mM, well above the 0.8 mM needed to saturate the rRNA promoter P1 in vitro. Many of the pools, including ATP, GTP, and UTP, begin to decrease while the cells are still in mid-log growth. After an almost universal drop in nucleotide concentration as the cells transition from logarithmic to stationary phase, there is a “rebound” of certain nucleotides, most notably ATP, after the cells enter stationary phase, followed by a progressive decrease. UTP, in contrast, increases as the cells transition into stationary phase. The higher UTP values might be related to elevated UDP-glucose/galactose, which was found to be at higher concentrations than expected in stationary phase. dTTP is the most abundant deoxynucleoside triphosphate (dNTP) in the cell despite the fact that its precursors, UDP and UTP, are not. All dNTPs decrease through the growth curve but do not have the abrupt drop, as seen with other nucleotides when the cells transition into stationary phase.     

Cyclic AMP and the control of aggregative phase gene expression in Dictyostelium discoideum.

The induction of aggregative phase functions and the acceleration of the onset of aggregation competence by nanomolar pulses of cyclic AMP can be mimicked by exposing developing cells to a high extracellular concentration of either cyclic AMP or cyclic GMP (5 × 10^?4M) during the first 1–2 hr of development. Pulses of cyclic AMP have previously been shown to result in oscillations of intracellular cyclic AMP concentration; we show that high extracellular concentrations of cyclic AMP and cyclic GMP cause intracellular cyclic AMP levels to increase. We describe a mutant, HM11, which has elevated levels of intracellular cyclic AMP from the beginning of development and which begins to accumulate cell-associated phosphodiesterase, an aggregative phase enzyme, within an hour of starvation. Our data suggest that the expression of aggregative phase functions is controlled by an elevation of intracellular cyclic AMP which may be either continuous or periodic.     

Production of carbohydrates by the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt. I. Effect of the concentration of nutrients in the culture medium

The chemical composition of cells of Chaetoceros affinis var. willei (Gran) Hustedt grown in batch culture was markedly influenced by the concentration of nutrients in the medium. In the logarithmic growth phase the content of cellular glucan was relatively low, but in the stationary phase the glucan content showed a rapid increase which seemed to coincide with the depletion of nitrate from the medium. This led to very pronounced variations in the ratio of protein to carbohydrate. This ratio can easily be determined and seems to be a sensitive and convenient parameter for characterizing the physiological state of the diatom 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.     

A natural CD label to probe the structure of the purple membrane from Halobacterium halobium by means of exciton coupling effects.

The polyamine and ribosome contents during the logarithmic phase of growth were much higher than those in the late logarithmic or stationary phase of growth. On the other hand, the Mg²⁺ content did not vary markedly throughout the different growth phases. These data, together with the results of a previous communication (1), suggest that increased polyamines during the logarithmic phase of growth may play significant roles not only in the neutralization of the negative charges of increased ribosomes but also in the increase of the velocity of polypeptide chain elongation by binding to the ribosomes. The polyphenylalanine synthetic activity of ribosomes from the logarithmic phase of growth was higher than that of ribosomes obtained from the stationary phase of growth in the presence of optimal concentrations of spermidine.