Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.     

Alkaline phosphatase production of Bacillus subtilis

Since alkaline phosphate activity increases in sporulation medium during the developmental period, in spite of the presence of inorganic phosphate, the uptake and intracellular concentration of phosphate were measured. While the uptake of inorganic phosphate decreases and the concentration of acid-soluble organic phosphate remains constant, the intracellular concentration of inorganic phosphate increases to about 30 mM after the end of growth. Some compound other than inorganic phosphate must therefore repress alkaline phosphatase. Other experiments showed that addition of glucose delays both the alkaline phosphatase increase and sporulation by about the same time.     

NUTRIENT-LIMITED GROWTH OF AUREOUMBRA LAGUNENSIS (PELAGOPHYCEAE), WITH IMPLICATIONS FOR ITS CAPABILITY TO OUTGROW OTHER PHYTOPLANKTON SPECIES IN PHOSPHATE-LIMITED ENVIRONMENTS

Growth and chemical compositional characteristics of the brown tide-forming alga, Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson, were studied through a series of nitrogen-limited and phosphate-limited continuous cultures over a range of growth rates. The specific growth rate of A. lagunensis was hyperbolically related to the cell quota of the limiting nutrient in ammonium-limited cultures. In phosphate-limited cultures, the relationship was best described by a straight line. The N cell quota of A. lagunensis ranges from about 20 fmol at zero growth rate under N-limited conditions to a high of roughly 85 fmol under N-replete conditions. Similarly, the P cell quota of A. lagunensis ranges from about 0.15 fmol at zero growth rate under P-limited conditions to a high of 2 fmol under P-replete conditions. Aureoumbra lagunensis has a very high N:P critical ratio (>100). The high N:P critical ratio, as well as the organism's apparent ability to use forms of phosphorus other than phosphate under severe phosphate deficiency, may partially explain its success in P-limited environments, such as the Laguna Madre. In addition, a uniqe quadratic relationship between the productivity index (PI) and growth rate was discovered. Such a relationship supports an earlier argument that PI may not be a good indicator of nutritional status.     

Overproduction of acetate kinase activates the phosphate regulon in the absence of the phoR and phoM functions in Escherichia coli.

A DNA fragment of Escherichia coli cloned on pBR322 elevated the production of alkaline phosphatase and phosphate-binding protein in a phoR phoM strain. Nucleotide sequence analysis and enzyme assays revealed that the DNA fragment contained the ackA gene, which codes for acetate kinase. A high gene dosage of ackA was needed to induce the production of alkaline phosphatase and phosphate-binding protein in this strain. Overexpression of ackA elevated the intracellular ATP concentration, an effect that might be related to activation of the phosphate regulon in the phoR phoM strain.     

Prevalence of a calcium‐based alkaline phosphatase associated with the marine cyanobacterium Prochlorococcus and other ocean bacteria

Phosphate plays a key role in regulating primary productivity in several regions of the world's oceans and here dissolved organic phosphate can be an important phosphate source. A key enzyme for utilizing dissolved organic phosphate is alkaline phosphatase and the phoA‐type of this enzyme has a zinc cofactor. As the dissolved zinc concentration is low in phosphate depleted environments, this has led to the hypothesis that some phytoplankton may be zinc‐P co‐limited. Recently, it was shown that many marine bacteria contain an alternative form of alkaline phosphatase called phoX, but it is unclear which marine lineages carry this enzyme. Here, we describe the occurrence in low phosphate environments of phoX that is associated with uncultured Prochlorococcus and SAR11 cells. Through heterologous expression, we demonstrate that phoX encodes an active phosphatase with a calcium cofactor. The enzyme also functions with magnesium and copper, whereas cobalt, manganese, nickel and zinc inhibit enzyme activity to various degrees. We also find that uncultured SAR11 cells and cyanophages contain a different alkaline phosphatase related to a variant present in several Prochlorococcus isolates. Overall, the results suggest that many bacterial lineages including Prochlorococcus and SAR11 may not be subject to zinc‐P co‐limitation.     

Nutrient Utilization Strategies of Algae and Bacteria after the Termination of Nutrient Amendment with Different Phosphorus Dosage: A Mesocosm Case

The impacts of nutrient amendment termination on the growth strategies of algae and bacteria were conducted in experimentally designed mesocosm in which two different phosphorus (P) dosages were treated. The algal community composition did not change greatly in Group A (low phosphorus) and Group B (high phosphorus). In Group A, the secretion of bacterial alkaline phosphatase (AP) after nutrient termination stimulated bacterial phosphorus acquisition, which caused the decrease in algal phosphorus levels, in terms of the increase of bacterial abundance and bacterial production, as well as the decrease in chlorophyll a and particulate organic carbon. The algal collapse resulted in dissolved organic carbon secretion, further fuelling bacterial growth. In Group B, excess phosphorus input urged algae to store phosphorus as poly-phosphate. When phosphorus input ceased, in order to maintain their used high phosphorus demand, algae strengthened to gain phosphorus through the hydrolysis of dissolved organic phosphorus in water column and ploy-phosphate inside the cells by AP, evidenced by high algal alkaline phosphatase activity, algal growth continuation, and bacterial growth decline. These facts indicated that phosphorus content should reduce to a lower level than expected, so that algal bloom can be effectively controlled in eutrophic water bodies.     

Alterations in Activity of Phosphatases during the Peridinium Bloom in Lake Kinneret

Acid and alkaline phosphatases have been isolated from Peridinium cinctum f. westii (Dinophyceae) during an algal bloom in Lake Kinneret. Acid phosphatase activity was fairly constant over the entire period of the bloom, although fluctuations in activity appeared to correlate with the chlorophyll content of the cells. Histochemical studies showed that the enzyme was localized inside the cell. Alkaline phosphatase activity was very low until May, a month after the peak of the bloom, when it increased sharply. Polyacrylamide gel electrophoresis revealed one or two bands of alkaline phosphatase that increased in intensity as the bloom progressed. However, the highest activity of the enzyme (in the last sample collected) corresponded to a new, very intense band on the gels. Similarly to acid phosphatase, alkaline phosphatase was also localized inside the cell. The appearance of alkaline phosphatase is probably related to the available phosphate concentration in the lake, although the influence of other factors that may contribute to the induction of the enzyme cannot be ruled out.     

THE IDENTIFICATION AND PURIFICATION OF A CELL-SURFACE ALKALINE PHOSPHATASE FROM THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

Two cell-surface proteins were identtjied in the dinoflagellate Prorocentrum minimum (Pavillard) Schilkr strain CCMP 1329 that are evident in phosphate-limited cultures, but not in nitrate-limited cultures or cultures growing exponentially in complete media. These proteins were detected by labeling cell-surface proteins with the biotinylating reagent succinimidyl 6-(biotinamido) hexanoate. One protein, of appoximately 200,000 daltons was purified using differential centrifugation, detergent extraction, and gel filtration chromatography. This purified protein was able to hydrolyze orthophosphate groups from p-nitrophenylphosphate at pH 8, indicating it is an alkaline phosphatase, although it is larger than other alkaline phosphatases isolated to date tom most microorganisms. This protein may be induced to help P. minimum cleave orthophosphate groups from organic forms of phosphate in marine environments. Ultimately, this protein could represent a unique antigen for developing an antibody probe for examining the relationships between phosphate stress and bloom formation in P. minimum, and perhaps other dinoflagellates, in the field.     

Phosphatases and the utilization of organic phosphorus by Rhizobium leguminosarum biovar viceae

Rhizobium leguminosarum biovar viceae strain TAL 1236 growing on different organic phosphorus compounds as sources of phosphate exhibited phosphatase activities. The strain was able to produce both acid and alkaline phosphatases. However, its ability to produce alkaline phosphatase was much higher. When cellular phosphate fell to 0.115% of cell protein, cellular and extracellular phosphatase activities were promoted. Mg²⁺, Co²⁺ and Ca²⁺ enhanced slightly the activity of alkaline phosphatase more than acid phosphatase. However, Mn²⁺ and Fe²⁺ activated acid phosphatase rather than alkaline phosphatase. It may be concluded that Rh. leguminosarum plays an important role in the release of phosphorus from its organic compounds through the action of phosphatases which can be slightly activated by a range of cations.     

Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase.

The effect of CO2+ on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelating agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 +/- 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium.     

Community development and modes of phosphorus utilization in a late summer ecosystem in the central Gulf of Finland, the Baltic Sea

The development of a filamentous, nitrogen-fixing cyanobacterial bloom was followed during July–August 1990 in a stratified basin in the central Gulf of Finland, Baltic Sea. Hydrography, dissolved inorganic, particulate and total nutrients, chlorophyll a, alkaline phosphatase activity, ³²PO₄-uptake and phytoplankton species were measured. The study period was characterized by wind-induced mixing events, followed by marked nutrient pulses and plankton community responses. Phosphate uptake was highest throughout the study period in the size fraction dominated by bacteria and picocyanobacteria (< 2 µm) and the proportion of uptake in the size fraction 2–10 µm remained low (2–6%). Higher phosphate turnover times were observed in a community showing signs of enhanced heterotrophic activity. The bloom of filamentous, nitrogen-fixing cyanobacteria Aphanizomenon flos-aquae was promoted by a nutrient pulse with an inorganic nutrient ratio (DIN:DIP) of 15. The results show that the quality, frequency and magnitude of the physically forced nutrient pulses have an important role in determining the relative share of the different modes of phosphorus utilization and hence in determining the cyanobacterial bloom intensity and species composition in the Baltic Sea.     

Effect of Aluminum Phosphate on Alkaline Phosphatase Activity of Polyurethane Foam Immobilized Cyanobacteria

The impact of insoluble phosphorus such as aluminum and rock phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria was assessed. Polyurethane foam immobilized Nodularia recorded the highest alkaline phosphatase activity of 9.04 (m. mol p-nitrophenol released h^–1 mg^–1 protein) in vitro. A higher concentration of aluminum phosphate was recorded a 25% reduction in alkaline phosphatase activity, ammonia content, and available phosphorus in culture filtrate of polyurethane foam immobilized cyanobacteria. In general, immobilized cyanobacteria exhibited a higher alkaline phosphatase activity in rock phosphate than aluminum phosphate.     

Activity of acid and alkaline phosphatases (intracellular and extracellular) in rhyzosphere fungi from Arachis hypogaea (Papiloneaceae)

The potential role of the fungi, isolated from the peanut rhizosphere, in the production of extracellular and intracellular acid and alkaline phosphatase, was evaluated in vitro. Acid and alkaline extracellular phosphatases showed the highest activities, and the Penicillium species were the most efficient in their production. The correlation analysis showed that extracellular acid and intracellular acid phosphatase produced by Aspergillus niger A. terreus, Penicillium sp. y P. brevicompactum were negatively correlated; while the extracellular and intracellular phosphatase activities, were positively correlated. The extracellular acid phosphatase activities produced in vitro by majority of fungi assayed, were not correlated with the acid phosphatase activity present in the peanut soil rhizosphere. Nevertheless, the extracellular alkaline phosphatase activities produced in vitro, were negatively correlated with the extracellular alkaline phosphatase activities present in the rhizosphere. The ability of phosphatase production by fungi isolated from peanut rhizosphere suggests they have great potential to contribute to the P mineralization in this zone.     

Seasonal variation in the phosphatase activity of waters and sewage sludges

Summary The alkaline phosphatase activity of water from eight locations differing in orthophosphate concentration has been determined over a period from late Autumn to late Summer. Evidence for induction/repression effects was conjectural, but cellular activity was highest in the environment of lowest orthophosphate concentration. Environments were sampled on a number of occasions and pH/phosphatase activity profiles constructed. The pH of maximum activity of low phosphate environments was in the acid region, that of high phosphate environments was in the alkaline region. There was little difference in the character and distribution of constitutive phosphatases in representative bacterial cultures from a high phosphate and a low phosphate environment. It seems likely that the phosphatase activity of a water at a particular time will be influenced by its nutrient and physico-chemical status as well as its ambient orthophosphate concentration.     

Inorganic phosphate transport in Escherichia coli: involvement of two genes which play a role in alkaline phosphatase regulation

Two classes of alkaline phosphatase constitutive mutations which comprise the original phoS locus (genes phoS and phoT) on the Escherichia coli genome have been implicated in the regulation of alkaline phosphatase synthesis. When these mutations were introduced into a strain dependent on a single system, the pst system, for inorganic phosphate (P(i)) transport, profound changes in P(i) transport were observed. The phoT mutations led to a complete P(i) (-) phenotype in this background, and no activity of the pst system could be detected. The introduction of the phoS mutations changed the specificity of the pst system so that arsenate became growth inhibitory. Changes in the phosphate source led to changes in the levels of constitutive alkaline phosphatase synthesis found in phoS and phoT mutants. When glucose-6-phosphate or l-alpha-glycerophosphate was supplied as the sole source of phosphate, phoT mutants showed a 3- to 15- fold reduction in constitutive alkaline phosphatase synthesis when compared to the maximal levels found in limiting P(i) media. However, these levels were still 100 times greater than the basal level of alkaline phosphatase synthesized in wild-type strains under these conditions. The phoS mutants showed only a two- to threefold reduction when grown with organic phosphate sources. The properties of the phoT mutants selected on the basis of constitutive alkaline phosphatase synthesis were similar in many respects to those of pst mutants selected for resistance to growth inhibition caused by arsenate. It is suggested that the phoS and phoT genes are primarily involved in P(i) transport and, as a result of this function, play a role in the regulation of alkaline phosphatase synthesis.     

PHOSPHORUS,NITROGEN AND THE GROWTH OF ALGAE IN LAKE KINNERET1

The intracellular concentrations of carbon, nitrogen, phosphorus and chlorophyll a of phytoplankton and zooplankton in Lake Kinneret were determined from 1969 to 1973. The ratios C:P, C:N, chlorophyll a:P, chlorophyll a:N of the algae showed fluctuations which could be related to the nutrient conditions that influence the annual pattern of phytoplankton development, especially in respect to the dinoflagellate bloom of Peridinium cinctum (OFM) Ehr. f. westii (Lemm.) Lef. Relatively high intracellurar P values at the start of the bloom indicated adequate availability of this nutrient and luxury consumption over a short period of time. Later, Peridinium continued to grow despite unusually high C:P ratios (> 300:1). In most years, phosphorus may have eventually limited growth, however, in 1970, the bloom censed despite comparatively high intracellular P concentrations. These observations, together with supplementary evidence from nutrient addition experiments and determinations of specific alkaline phosphatase levels, indicated that, for most of the growth phase of the bloom, Peridinium cells were not directly limited by P. The decline of the bloom usually, but not always (e.g., in 1970), was marked by very high C:P ratios. Thus, a shortage of P may often be a contributory factor to the cessation of the Peridinium bloom and may be limiting phytoplankton growth in the fall. Over the years 1969–73, possibly due to an overall drop in salinity, there appears to be a trend to lower levels of biologically bound phosphorus in Lake Kinneret, without a concomitant decrease in carbon biomass.     

Phosphorylation of the growth factors bFGF, NGF and BDNF: a prerequisite for their biological activity

The aim of this work was to test whether growth factors such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) undergo autophosphorylation and whether this affects their biological activity. Incubation of those growth factors with [gamma-(32)P]ATP resulted in phosphorylation in vitro. The phosphate bond was resistant to alkaline pH, yet acid-labile. Addition of alkaline phosphatase resulted in time and protein dependent dephosphorylation. Concomitantly, alkaline phosphatase abolished the neuroprotective effect of those growth factors upon oxygen and glucose deprivation and upon staurosporine-induced cell death. For those studies, we were using primary cultures of cortical and hippocampal neurons from embryonic and neonatal rats. Incubation of bFGF with non-hydrolyzable ATP-gammaS resulted in phosphorylation and in neuroprotection resistant to alkaline phosphatase. We conclude that bFGF, NGF and BDNF undergo autophosphorylation on site(s) other than serine, threonine, tyrosine and/or ATP-binding, and that this binding of phosphate is essential for neuroprotection in vivo.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.