31P-NMR DIFFERENTIATION BETWEEN INTRACELLULAR PHOSPHATE POOLS IN COSMARIUM (CHLOROPHYTA)1

³¹P nuclear magnetic resonance (NMR) spectroscopy of intact Cosmarium sp. cells is presented as a suitable tool for the differentiation of intracellular accumulation pools of polyphosphates. The cold trichloroacetic acid (TCA) insoluble fraction is shown to contain most of the total cellular phosphate in the phosphate rich Cosmarium cells. Moreover, evidence from a ³¹ P-NMR study and electron microscopic observations of cold TCA treated Cosmarium cells indicate that this fraction consists mostly of polyphosphates which seem to retain the native morphological structure observed in the untreated cells. The determination of orthophosphate in the hot water extract of Cosmarium cells did not measure the polyphosphate pools. Determination of total phosphorus content in the hot water extract rendered a value three times higher than the frequently used orthophosphate determination procedure. However, as revealed by the ³¹P-NMR spectra and the chemical analyses of the extract and of the treated cells, even total phosphorus in the extract measured only 30% of the total cellular phosphorus. ³¹P-NMR enabled the unequivocal chemical identification of the major phosphate compounds in the hot water extract (“Surplus P”) as orthophosphate and polyphosphates of about 10 phosphate units chainlength. More than 70% of the accumulation pool of polyphosphates was still in the cells after extraction. However, the electron microscopy study revealed that the native granular structure of polyphosphates had been destroyed by the hot water extraction procedure.     

Relationship of Cellular Energetics to RNA Metabolism in Tetrahymena pyriformis W.*

Cultures of Tetrahymena pyriformis in a non-nutrient buffer degrade RNA and excrete hypoxanthine, uracil and orthophosphate. Glucose addition leads to the retention of a portion of the purine, pyrimidine, and orthophosphate by the cells; however, the hexose has little influence on the RNA level. Acetate supplementation has no effect on RNA degradation or on the distribution of the catabolic products between the cells and the environment. Interruption of oxidative phosphorylation by 2,4-dinitrophenol results in an increase in RNA degradation. This action is annulled by the glycolytic substrate, glucose, but not by acetate. A combination of iodoacetic acid and glucose blocks glycolysis and increases cellular RNA loss which can be reversed by the addition of the citric acid cycle substrate, acetate. These findings suggest that the available cellular energy supply in starved cells is sufficient to regulate the rate of RNA degradation. Disruption of ATP generation by the appropriate inhibitors, however, allows the demonstration of the importance of energy-yielding reactions in the determination of the amount of nucleic acid loss. It appears that glycolysis and oxidative phosphorylation are equally efficient in sustaining the regulatory process. RNA synthesis during starvation conditions is a discontinuous process with a sharp rate change after 30 min of incubation. 2,4-Dinitrophenol inhibits [2-¹⁴C] uracil incorporation into the nucleic acid. Glucose does not annul the inhibition of synthesis in contrast to the influence of the hexose on RNA degradation. This observation demonstrates that the synthetic and degradative processes are not directly coupled. Glycogen synthesis and RNA degradation appear to compete for the available energy supply and respond in a similar fashion to the metabolic inhibitors and carbon sources.     

Influence of ions and tonicity of RNA metabolism in Tetrahymena pyriformis

Net RNA degradation occurs in Tetrahymem pyrifmmis when this ciliate is suspended in a non-nutrient medium. The quantity and quality of the excretion products is at least partially under the control of the ionic content and the tonicity of the cellular environment. The excretion of ultraviolet-absorbing materials was found to be elevated by sodium ions in a medium isotonic to the culture fluid, or by a hypertonic environment. Magnesium counteracted these effects. In isotonic suspension, sodium and magnesium ions lowered orthophosphate excretion; however, sodium altered the nature of the phosphate products so that acidlabile phosphates were also excreted rather than solely orthophosphate. Similar results were obtained in a hypertonic environment with or without sodium. The degree of purine and pyrimidine loss from the cells in all conditions of suspension was reflected in the amount of RNA degraded. The ion and tonicity effects apparently reflect events which alter the stability of the RNA and the properties of the membrane system, resulting in changes in both the rate of RNA degradation and the nature of the excreted products. The rates of orthophosphate excretion appear to be affected by changes in the acid-base balance within the cell which may be governed by the cation levels. The manipulation of the ionic content and tonicity of the medium offers a convenient method for obtaining cells reduced in RNA content.     

THE DISTRIBUTION OF THE WATER-SOLUBLE INORGANIC ORTHOPHOSPHATE IONS WITHIN THE CELL: ACCUMULATION IN THE NUCLEUS : Electron Probe Microanalysis

Lead acetate treatment of unfixed cells immobilizes the intracellular water-soluble, inorganic orthophosphate ions as microcrystalline lead hydroxyapatite precipitates (see reference 1). These precipitates have been analyzed with the electron microprobe. A much higher concentration of phosphorus has been found in the nucleoli of maize root tip cells fixed in lead acetate-glutaraldehyde (organic phosphorus plus inorganic orthophosphate), as compared to the nucleoli of roots fixed in glutaraldehyde alone (organic phosphorus). The concentration of the inorganic orthophosphate pool in these nucleoli is three to five times as high as the concentration of the macromolecular organic phosphate. Since nearly all of the latter is in RNA, the concentration of inorganic phosphate in the nucleolus is calculated to be roughly 0.5–0.8 M. About 30%—and up to 50%—of the total cellular inorganic phosphate is accumulated in the nucleolus since the mean concentration per cell is about 10^-2 M. In the extranucleolar part of the nucleus the mean concentration was estimated by densitometry to be roughly six times less than in the nucleolus (⩽ 0.1 M), and appears more concentrated in the nucleoplasm than in the condensed chromatin. While there is no direct evidence for the concentration in the cytoplasm, it certainly must be much lower than the mean cellular level (i.e., < 10^-2 M) since the nucleus is about 10% of the total cell volume. The implications of this compartmentation in the intact cell are discussed in connection with (A) the availability of orthophosphate ions for the cytoplasm in those processes in which these ions affect the rate of enzymatic reactions, and (B) protein nucleic acid interactions within the nucleus and nucleolus.     

Divalent cations and inorganic phosphate metabolism in starved Tetrahymena

Tetrahymena pyriformis maintained under starvation conditions release orthophosphate into the suspension medium. Ca²⁺ and/or Mg²⁺ addition reduced the amount of orthophosphate excreted during a 3-h period. Cellular orthophosphate levels were not altered by divalent cation supplements; however, an increase in the pyrophosphate content was observed which was equivalent in amount to the reduction in orthophosphate efflux. These observations suggest that divalent cations are important not only in the acquisition of phosphorus during growth but also in the conservation of this element during starvation.     

Mechanism of Purine Arabinoside Synthesis by Bacterial Transarabinosylation Reaction

The mechanism of purine arabinoside synthesis from uracil arabinoside and purine bases via the bacterial transarabinosylation reaction was investigated. Arabinose-1-phosphate was isolated from the reaction mixture in the form of the barium salt and proved to be the intermediate of the reaction. Two enzyme fractions were obtained from Enterobacter aerogenes by means of heat treatment, ammonium sulfate fractionation and DEAE-cellulose column chromatography. One enzyme split uracil arabinoside into uracil and arabinose-1-phosphate in the presence of inorganic phosphate and the other synthesized hypoxanthine arabinoside from arabinose-1-phosphate and hypoxanthine. The substrate specificity of these enzymes indicated that the former was uridine phosphorylase and the latter was purine nucleoside phosphorylase, respectively. Hypoxanthine arabinoside was synthesized from uracil arabinoside and hypoxanthine only in the presence of both enzymes and inorganic phosphate.     

Uracil metabolism in golden hamster after irradiation.

The catabolism of uracil and the total balance of excreted radioactivity were studied in golden hamsters after a peroral application of 14C-uracil. Twenty-four hours after administration most of the radioactivity taken up appeared in expired carbon dioxide. The percent proportion of radioactivity in carbon dioxide was independent of the amount of uracil administered. On the other hand, the percentage of radioactivity excreted in urine depended on the amount of uracil taken up, high doses of the compound causing up to eight-fold increase in urine-excreted radioactivity. Most of the exogenously-administered uracil was catabolized within the first 5 hours. Irradiation had no substantial effect on the dynamics of uracil catabolism. Analysis of urine revealed that most urine-excreted radioactivity is in the form of uracil. On peroral application of high doses of uracil to irradiated hamsters, their urine was found to contain barbituric acid which originated from uracil.     

Limax Amoebae in Public Swimming Pools of Albany, Schenectady, and Rensselaer Counties, New York: Their Concentration, Correlations, and Significance

A survey was conducted on 30 halogenated public swimming pools, located in Albany, Schenectady, and Rensselaer counties, to determine their open-water limax amoeba densities. Six were outdoor pools. Other variables measured were the standard plate count, total seston, free residual chlorine or bromine, total alkalinity, total hardness, orthophosphate, total soluble phosphorus, specific conductance, pH, temperature, and several engineering parameters including the rate and type of filtration as well as a saturation index. Amoebae were isolated on agar plates at 37°C using heat-killed bacterial suspensions of Enterobacter cloacae or Escherichia coli. Most probable number estimates of amoebic densities ranged from not detectable (<0.01) to 110 amoebae per liter. The median concentration of amoebae was 0.9/liter. Eighty percent of the pools examined had less than 5 amoebae per liter. Significant correlations (P < 0.05) were found between amoebic densities and the log₁₀ of the standard plate count, orthophosphate, and total soluble phosphorus. No significant difference was found between amoebic densities in outdoor and indoor pools. Preliminary tests for the presence of the human pathogen Naegleria fowleri were inconclusive.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.     

Intracellular and Extracellular Nucleotides and Related Compounds During the Development of Myxococcus xanthus

Changes in nucleotide pools and extracellular nucleotides during the developmental cycle of the myxobacterium Myxococcus xanthus were determined using a high-pressure liquid chromatography nucleotide analyzer. A general increase in all nucleotide pools occurred during the morphological phase of glycerol conversion of vegetative cells to myxospores. The levels of the nucleoside triphosphate pools remained high as the myxospore matured and throughout subsequent germination. Oxidized nicotinamide adenine dinucleotide levels were elevated in the dormant myxospore and then declined during germination. The adenylate energy charge value was 0.85 +/- 0.02 for vegetative cells, germinating myxospores, and 6-h-old myxospores. It was interesting that the value for the so-called dormant myxospore was the same as that characteristic of physiologically active cells. The germinating myxospores excreted large quantities of uracil along with lesser quantities of purine nucleoside monophosphates. Although the source of the extracellular uracil cannot be determined from these experiments, it may have been derived from a shift in base ratios accompanying an assumed ribonucleic acid turnover during germination.     

不同磷浓度植株残体降解对紫色土磷素有效性的影响

植株残体降解可直接或间接地影响土壤磷素的有效性,为探讨不同磷浓度植株残体降解对紫色土磷分级体系的影响,结合31P核磁共振分析技术,选取了3种磷浓度不同的植物残体与两种紫色土进行室内模拟培养试验,得出了以下研究结论:(1)添加植株残体显著增强了紫色土呼吸强度,且紫色土分级体系中的活性磷含量均高于对照处理(2)31P-NMR分析结果得知,植株残体的正磷酸盐、磷酸单酯占浓缩液全磷比例的90%以上,高磷植株的正磷酸盐和磷酸单酯含量显著高于中磷和低磷植株,土壤磷素有效性的变化与植株残体的正磷酸盐和磷酸单酯含量有关;(3)紫色土分级体系中的活性磷在0 d含量最高,随着培养周期的延长,土壤磷素有效性会出现降低的趋势;酸性紫色土的累积呼吸强度、分级体系中活性磷(Resin-P、Na HCO3-Pt)所占比例均高于中性紫色土,与土壤钙含量有关。综上所述,植株残体的磷浓度越高,更有利于提高土壤磷素的有效性,本研究结果为农业生产中秸秆还田技术提供了理论参考。     

Studies on the Autolysis of Aspergillus oryzae

The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.

In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.

Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases.     

Limnology of Shagawa Lake,Minnesota, prior to reduction of phosphorus loading

Various limnological parameters have been measured in Shagawa Lake, a culturally eutrophic lake in northeastern Minnesota, from 1970–1972. These included temperature; specific conductance; dissolved oxygen; alkalinity; pH; total and orthophosphate phosphorus; nitrate, nitrite and ammonia nitrogen; chlorophyll a; transparency; major cations; and selected trace elements. The lake exhibited many characteristics of high productivity. Concentrations of chlorophyll a reached 60 µg/l during summer months and were reflected in pH values in excess of 9. Anaerobic conditions developed during both summer and winter and, during these intervals, large pools of available nutrients developed in the lower waters. Manganese and iron concentrations varied greatly as a consequence of changes in the oxygen regime. Major cations and other trace elements showed no yearly pattern.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (P_i) level. In contrast, normal cells excreted nucleosides only at low P_i level while at high P_i levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.     

The Effect of Grazing by a Ciliated Protozoan on Phosphorus Limitation of Heterotrophic Bacteria in Batch Culture1

ABSTRACT. The yield of the bacterium Enterobacter aerogenes and the ciliate Colpidium colpoda was dependent on initial phosphorus concentrations in batch cultures containing 125 or 250 mg/liter glutamate and 50–1000 μg/liter phosphorus. For both, yield per unit phosphorus declined at higher phosphorus concentrations. A marked decline in growth rate in bacterial cultures was coincident with the depletion of dissolved phosphorus and the development of rapid orthophosphate turnover times. Colpidium introduced to these cultures consumed about 16,000 bacteria/h/ciliate while multiplying exponentially and relieved phosphorus limitation, as indicated by a longer turnover-time for phosphate. The longer turnover-time was due to the reduction of bacterial numbers; in cultures with ciliates, bacteria appear to be more active in taking up phosphate, and much of the total phosphorus accumulates in ciliates. Ciliates released both inorganic and organic phosphorus, but the organic phosphorus did not accumulate to excess in the cultures to an extent that would indicate that it is less used by bacteria. Although ciliates release enough phosphorus to account for ca. 20% of the bacterial uptake, ciliates appear to behave as phosphorus sinks as much as phosphorus recyclers in these closed systems.     

Testing the growth rate and translation compensation hypotheses in marine bacterioplankton

Two different hypotheses have been raised as to how temperature affects resource allocation in microorganisms. The translation-compensation hypothesis (TCH) predicts that the increase in enzymatic efficiency with temperature results in fewer required ribosomes per cell and lower RNA:protein ratio. In contrast, the growth rate hypothesis (GRH) predicts that increasing the growth rate with temperature requires more ribosomes and hence a higher cellular RNA:protein. We tested these two hypotheses in laboratory cultures of Prochlorococcus and Alteromonas as well as over an annual cycle in the Eastern Mediterranean Sea. The RNA:protein of Alteromonas mostly decreased with temperature in accordance with the TCH, while that of Prochlorococcus increased with temperature, as predicted by the GRH. No support was found for either hypothesis in surface waters from the Eastern Mediterranean, whereas the fraction of phosphorus in RNA was positively correlated with per-cell bacterial production in the deep chlorophyll maximum, supporting the GRH in this niche. A considerable part of the cellular phosphorus was not allocated to RNA, DNA, phospholipids or polyphosphate, raising the question which cellular molecules contain these P reserves. While macromolecular quotas differed significantly between laboratory cultures and field samples, these were connected through a power law, suggesting common rules of resource allocation.     

Pool Control of Uracil Uptake by Synchronous Chlorella

Experiments with autospores of Chlorella fusca showed that the uptake rate of uracil was reduced as much as 40 % as the result of formation of soluble pools of uracil and its metabolites from exogenous uracil. Using synchronous cultures similar results were obtained with cells at other developmental stages.     

Regulation of Phosphate Accumulation in the Unicellular Cyanobacterium Synechococcus

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acid-insoluble polyphosphate were lowered in Synechococcus in proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10% of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparent K(m) of about 0.5 muM, but V(max) was 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 15 degrees C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity.