Intracellular phosphorus pool of the cyanobacteriumSpirulina platensis

An intracellular phosphorus pool in a monoculture of the cyanobacteriumSpirulina platensis was assessed using radioactive and nonradioactive phosphorus. The derived dependence of specific growth rate on the intracellular content of mineral phosphorus can be presented in the form of the Droop equation. It was found that the stage of replenishment of the intracellular phosphorus pool may affect the phosphorus turnover estimation in aquatic environments from the results of short-term measurements of phosphorus uptake     

Phosphorus cyeling in aquatic microorganisms studied by phased uptake of33P and32P

An isotopic technique using³²P and³³P simultaneously was developed to examine cellular and intracellular phosphorus cycling in microorganisms. The method was tested with cultures of two algae (Ankistrodesmus sp. andDunaliella tertiolecta) and a marine bacterium (Beneckea natriegens). Intact cells and intracellular fractions showed phosphorus cycling, with the fastest exchange rates being observed in the pool extracted by hot water. The technique should also be applicable to cell and tissue cultures or simple microcosms.     

Nucleic acid economy in bacteria infected with bacteriophage T2

1. During the first 10 minutes of viral growth following infection of E. coli by phage T2 in broth, a pool of DNA is built up that contains phosphorus later to be incorporated into phage. This pool receives phosphorus from, but does not contain, the bacterial DNA. 2. After 10 minutes, DNA synthesis and phage maturation keep pace in such a way that the amount of precursor DNA increases moderately for a time and then remains constant. 3. The pool so described is defined in terms of the kinetics of transport of phosphorus from its origins in the culture medium, the bacterial DNA, and the DNA of the parental phage, to the viral progeny. The most interesting parameter of this system is the size of the precursor pool, which measures 10^–9 to 2 x 10^–9 µg. DNA-P (50 to 100 phage particle equivalents) per bacterium. 4. Neither the precursor nor the intracellular phage population exchanges phosphorus with the phosphate in the medium. More interestingly, the phosphorus in mature phage does not exchange with phosphorus in the precursor, showing that maturation is an irreversible process. 5. Maturation is also a remarkably efficient process. About 90 per cent of labeled phosphorus introduced early into the precursor pool is later incorporated into phage. 6. Viral DNA is synthesized at the rate of about 1.5 x 10^–10 µg. DNA-P (7 or 8 phage particles) per bacterium per minute. This is somewhat faster than bacterial DNA is formed, but considerably slower than RNA is formed, in uninfected bacteria. 7. The transport of phosphorus from medium to viral precursor DNA takes an average of 8 or 9 minutes, and from precursor to phage an additional 7 or 8 minutes. 8. Metabolically active RNA has been detected in infected bacteria.     

Microbial Phospholipid Synthesis as a Marker for Microbial Protein Synthesis in the Rumen

Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with (33)P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of (33)P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with (33)P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs.     

我国北方3种典型土壤-作物体系中微生物量磷库特征

土壤微生物量磷(Microbial Biomass Phosphorus, MBP)是土壤磷组分中最为活跃的形态,在土壤磷素的形态转化与生物地球化学循环过程中起着关键作用,是植物可利用磷的重要来源。研究土壤MBP库容的大小对于充分认识微生物的固磷潜力和掌握土壤磷素循环与转化能力意义重大。以我国北方农田3种典型的土壤-作物体系为研究对象,基于定点采样,通过分析测定采集的362个表层(0—30 cm)土壤样品来量化不同土壤-作物体系MBP库容的大小。结果表明：黑土-春玉米、潮土-冬小麦/夏玉米、灰漠土-棉花体系表层土壤MBP平均含量分别为17.36、14.45、8.75 mg/kg,且不同土壤-作物体系间MBP含量存在显著差异;3种土壤-作物体系表层土壤(0—30 cm)MBP库容的大小分别为83.60、54.26、39.80 kg P/hm~2,其储存的磷在数量上相当于当季作物需磷量的1.10—2.73倍,表明土壤MBP库是农田生态系统中一个不容忽视的巨大有效养分磷储库。其库容的大小受土壤性质和气候因素的共同影响,土壤pH、有机碳、年均气温和年均降雨量是我国北方农田土壤MBP库容大小的主...     

In vitro construction of the tufB-lacZ fusion: analysis of the regulatory mechanism of tufB promoter

Summary Genetic studies suggest that the so-called phosphorus-family of enzymes inN. crassa are controlled by a complex system of regulatory genes which are responsive to the level of phosphorus in the growth medium. The intracellular metabolite(s) that interact with this system to signal changes in the external phosphorus concentration has not been identified. In this study the pools of acid-soluble, phosphorus-containing, compounds are measured in wild-type and phosphorus-family enzyme regulatory mutant strains ofN. crassa before and during phosphorus starvation.Prolonged phosphorus starvation of wild-typeN. crassa failed to alter significantly the pre-starvation level of intracellular orthophosphate, suggesting that intracellular P_i would be a poor effector signal for the control of the phosphorus family enzymes. However, inorganic pyrophosphate (PP_i) decreased 15-fold, and tri- and tetrapolyphosphate (PPP_i and PPPP_i) increased 3- to 5-fold within 15 minutes after transfer of the wild-type strain to phosphorus-free medium. Phosphate starvation of seven different regulatory gene mutant strains resulted in a rapid decrease in the PP_i pool similar to that which occurred in the wild-type. However, only two of these seven strains showed increased PPP_i and PPPP_i pools following phosphate starvation. Additional experiments demonstrated that PP_i pools, but not PPP_i and PPPP_i pools, were unaffected by several starvation regimens other than phosphorus starvation. Metabolic studies employing H₃ ³²PO₄ showed that the pool of PP_i was labeled to steady-state levels after two minutes of continuous labeling of a phosphate-sufficient culture. Furthermore, long-term steady-state labeling showed that the intracellular PP_i pool was directly responsive to the decrease in the extracellular P_i concentration of the medium resulting from cell growth. Growth on phosphoethanolamine, a phosphorus source that allows a modest degree of derepression even in growing cells, resulted in lower levels of PP_i than were seen in phosphate-grown cells. These observations suggest that PP_i may be involved in the mechanism responsible for the control of phosphorus-family enzyme regulatory gene product activity.     

Glucose Metabolism and Kinetics of Phosphorus Removal by the Fermentative Bacterium Microlunatus phosphovorus

Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch reactor to study the kinetics of uptake and release of extracellular compounds, in combination with ³¹P and ¹³C nuclear magnetic resonance (NMR) to characterize intracellular pools and to trace the fate of carbon substrates through the anaerobic and aerobic cycles. The organism was subjected to repetitive anaerobic and aerobic cycles to induce phosphorus release and uptake in a sequencial batch reactor; an ultrafiltration membrane module was required since cell suspensions did not sediment. M. phosphovorus fermented glucose to acetate via an Embden-Meyerhof pathway but was unable to grow under anaerobic conditions. A remarkable time shift was observed between the uptake of glucose and excretion of acetate, resulting in an intracellular accumulation of acetate. The acetate produced was oxidized in the subsequent aerobic stage. Very high phosphorus release and uptake rates were measured, 3.34 mmol g of cell⁻¹ h⁻¹ and 1.56 mmol g of cell⁻¹ h⁻¹, respectively, values only comparable with those determined in activated sludge. In the aerobic period, growth was strictly dependent on the availability of external phosphate. Natural abundance ¹³C NMR showed the presence of reserves of glutamate and trehalose in cell suspensions. Unexpectedly, [1-¹³C]glucose was not significantly channeled to the synthesis of internal reserves in the anaerobic phase, and acetate was not during the aerobic stage, although the glutamate pool became labeled via the exchange with intermediates of the tricarboxylic acid cycle at the level of glutamate dehydrogenase. The intracellular pool of glutamate increased under anaerobic conditions and decreased during the aerobic period. The contribution of M. phosphovorus for phosphorus removal in wastewater treatment plants is discussed on the basis of the metabolic features disclosed by this study.     

Folate utilization in Friend erythroleukemia cells

In order to study the generation, factors controlling endogenous folate pools, and their functional importance, Friend erythroleukemia cells were grown in media containing 100; 1,000; and 10,000 ng/ml of tritiated pteroylglutamic acid (3H)PteGlu1 and then studied in unlabeled media with varying amounts of PteGlu1. The intracellular folate pool was directly proportional to the PteGlu1 in which the cells were incubated. At equilibrium, greater than 95% of the labeled intracellular folate pool chromatographed as polyglutamyl folate, regardless of the exogenous folate concentration. The functional importance of the intracellular folate pool was studied by varying the endogenous pool and the exogenous (media) supply. The ability of the cells to replicate in the absence of exogenous folate was directly proportional to the intracellular polyglutamyl folate pool. The maximal rate of replication, however, required exogenous PteGlu1 in addition. The cell doubling time was the most important determinant of intracellular folate turnover; changes in the intracellular pool size and the extracellular folate concentration had no effect on the turnover time. In a rapidly proliferating tissue, the onset of functional folate deficiency will be determined by dilution of intracellular polyglutamates among progeny until a critical level is reached.     

Sodium transport and phosphorus metabolism in sodium-loaded yeast: simultaneous observation with sodium-23 and phosphorus-31 NMR spectroscopy in vivo

Simultaneous 23Na and 31P NMR spectra were obtained from a number of yeast suspensions. Prior to NMR spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they had no polyphosphate species visible in the 31P NMR spectrum. In the NMR experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na+, and a shift reagent for the Na+ NMR signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The NMR spectra revealed that the cells always lost Na+ and gained phosphorus. However, the nature of the Na+ efflux time course and the P metabolism differed depending on the medium. The Na+ efflux usually proceeded linearly until the amount of Na+ extruded roughly equalled the amount of NH4+ and orthophosphate initially present in the medium (external phosphate was added as NH4H2PO4). Thus, we presume this first phase reflects a Na+ for NH4+ exchange. The Na+ efflux then entered a transition phase, either slowing, ceasing, or transiently reversing, before resuming at about the same value as that of the first phase. We presume that this last phase involves the simultaneous extrusion of intracellular anions as reported in the literature. The phosphorus metabolism was much more varied. In the absence of exogenous glucose, the P taken up accumulated first as intracellular inorganic phosphate; otherwise, it accumulated first in the "sugar phosphate" pool. In most cases, at least some of the P left the sugar phosphate pool and entered the polyphosphate reservoir in the vacuole. However, this never happened until the phase probably representing Na+ for NH4+ exchange was completed, and the P in the polyphosphate pool never remained there permanently but always eventually reverted back to the sugar phosphate pool. These changes are interpreted in terms of hierarchical energy demands on the cells under the different conditions. In particular, the energy for the Na+ for NH4+ exchange takes precedence over that required to produce and store polyphosphate. This conclusion is supported by the fact that when the cells are "forced" to exchange K+, as well as NH4+, for Na+ (by the addition of 5 times as much K+ to the NH4+-containing medium), polyphosphates are never significantly formed, and the initial linear Na+ efflux phase persists possibly 6 times as long.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aspects of the phosphorus cycle in Hartbeespoort Dam (South Africa) Phosphorus loading and seasonal distribution of phosphorus in the reservoir

Hartbeespoort Dam, a hypertrophic, warm monomictic impoundment in South Africa, receives extremely high phosphorus loads (14.6–25.9 g m^–2 a^–1) that are dominated by point source discharges from municipal wastewater treatment works. The reduced state of the phosphorus discharged from the works has led to the dominance of the dissolved phosphorus pool by low molecular weight orthophosphates which are analytically detectable as soluble reactive phosphorus (SRP; 60% of total phosphorus pool). Seasonality in the in-lake total phosphorus pool is regulated by a combination of abiotic and hydrological processes; biotic processes appear to play a minor role. Mass balance calculations indicate that between 62 and 77% of the annual total phosphorus inflow load is retained within the impoundment each year.     

Alterations of the composition and size of the free fatty acid pool of Tetrahymena responding to low-temperature stress

Cells of Tetrahymena mimbres (formerly T. pyriformis NT-1) in midlogarithmic growth under isothermal conditions (at 39 degrees C) contained a very small, compositionally discrete pool of free fatty acids, principally (60.6% of the total free fatty acid mass) palmitic and stearic acids. The composition, degree of unsaturation, and size of this free fatty acid pool were rapidly (15 min or less) altered in response to chilling. During the acclimation period following chilling to 15 degrees C, the size of the free fatty acid pool increased from a mean value of 15.5 nmol free fatty acid/mumol lipid phosphorus in 39 degrees C cells to 24.2 nmol free fatty acid/mumol lipid phosphorus. The degree of free fatty acid saturation rapidly increased over the initial hour following the onset of hypothermal conditions, but by 24 h the unsaturated free fatty acid/saturated free fatty acid ratio was 0.35 (equivalent to a 2.7-fold increase in unsaturation relative to 39 degrees C controls (unsaturated/saturated ratio = 0.13) and 4.4-fold greater than cells acclimated for 1 h (unsaturated/saturated ratio = 0.08)). By 24 h the percentage of palmitic and stearic acids had decreased to 45.6%. Similar, and in some instances more pronounced, changes were observed to occur in triacylglycerol-bound fatty acids. Modulation of steady-state free fatty acid composition could also be achieved by the addition of exogenous fatty acids to the growth medium. The ability to manipulate the level of intracellular free fatty acids should prove to be a valuable experimental tool in determining how specific fatty acids regulate various lipid-modifying enzymes.     

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0?×?10⁶ cells mL^?1. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1 % was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L^?1 day^?1, starch concentration of 1.6 g L^?1, and starch productivity of 0.30 g L^?1 day^?1 were all obtained in the culture with the ICD of 3.0?×?10⁶ cells mL^?1. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH₂PO₄ was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.     

Physiological and structural effects of phosphorus starvation on the unicellular green alga Scenedesmus

The effects of phosphorus starvation on morphology and intracellular structure and on reactions related to the energy metabolism of the unicellular green alga Scenedesmus obtusiusculus (Chod.) were studied over a period of 96 h by employing transmission electron microscopy and various methods for measurement of physiological reactions. Increase in cell size and shape and in cell wall thickness are dominating features of phosphorus starvation. There is also an increase in number and size of starch granules and lipid globules and the internal structure of the cells appears successively disorganized. Shortage of phosphorus in the medium initially induces an increase of the adenylate pool whereas the energy charge value remains the same as for the controls. The photosynthetic and respiratory activities are high during incipient phosphorus starvation. After 24 h, as shortage of phosphorus becomes critical, the internal phosphorus reaches a low steady-state value, and this is also true for the adenylate energy charge. The total content of adenylates, however, peaks after 24 h of starvation and then decreases with increasing length of phosphorus starvation. Light-induced oxygen evolution appears not to be as much inhibited by a low phosphorus content in the cells as by the concomitant starch accumulation. The data indicate that the strategy for survival of the cells in a phosphorus-poor environment includes morphological and physiological changes that facilitate the transfer and adaption of the cells to environments with a more favourable supply of phosphorus, such as the often oxygen-poor but phosphorus-rich bottom zones.     

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I)-stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I)-stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.     

Kinetics of phosphorus absorption by<Emphasis Type="Italic">Corynebacterium bovis</Emphasis>

The initial rate of phosphorus uptake by phosphorus-limited cells ofCorynebacterium bovis grown in batch culture and in a chemostat was measured with [³²P] orthophosphate. It was dependent on the external phosphorus concentration and was inversely related to the amount of intracellular phosphorus. The relationship between the initial rate of uptake, intracellular phosphorus, and phosphorus concentration in the medium can be expressed in terms of Haldane's modification of the Michaelis-Menten equation.     

Kinetics of phosphorus absorption byCorynebacterium bovis

The initial rate of phosphorus uptake by phosphorus-limited cells ofCorynebacterium bovis grown in batch culture and in a chemostat was measured with [³²P] orthophosphate. It was dependent on the external phosphorus concentration and was inversely related to the amount of intracellular phosphorus. The relationship between the initial rate of uptake, intracellular phosphorus, and phosphorus concentration in the medium can be expressed in terms of Haldane's modification of the Michaelis-Menten equation.     

The role of phosphatases in the metabolism of Peridinium cinctum,from Lake Kinneret

The annual algal bloom (February–June) in Lake Kinneret consists almost entirely of the dinoflagellatePeridinium cinctum f.westii (Dinophyceae). To clarify the role of phosphatases in the alga, experiments were carried out using cells from culture or from the lake. In culture, as the external ambient orthophosphate (Pi) concentration decreased, alkaline phosphatase activity increased (and to some extent acid phosphatase activity, as well). Hot water extractable P decreased, although molybdate reactive phosphorus (MRP) appeared to be utilized in preference to the non-MRP component of this pool. Alkaline phosphatase inPeridinium collected from the lake as well as cells grown in culture under a high (3–6 mg l^–1) ambient Pi concentration in both continuous light and a 12:12 light-dark cycle, showed a diurnal fluctuation in activity. These results, together with previous observations suggest that the phosphatases inPeridinium are controlled by changes in intracellular phosphorus levels (other than the hot water extractable pool) and/or by other metabolic processes not directly involved in P nutrition.     

THE NATURE OF THE AMINO ACID POOL USED FOR PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The incorporation into brain slice protein of externally provided [1-¹⁴C]valine was measured at varying levels of valine in the medium, under conditions of constant protein synthesis and equilibration of intracellular valine specific activity. The results indicate that the valine pool used for protein synthesis is not identical to the pool of total free valine. Neither does the incorporation solely occur from an extracellular pool which is in equilibrium with the incubation medium. The data are compatible with a two-site activation model in which aminoacylation of tRNA occurs at both an internal site utilizing amino acid from the intracellular pool and an external (possibly membranous) site converting extracellular valine directly to valyl-tRNA. A good fit to the experimental observations is also provided by a compartmented intracellular valine pool model.     

Compartmentation of free amino acids for protein synthesis in rat liver

The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [(3)H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2-3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.