A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)     

Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae

The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.     

Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications

BACKGROUND: The fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF(R)-97 phosphate, for Enzyme-Labeled Fluorescence) has been used primarily in microscope-based imaging applications to detect endogenous AP activity, antigens and various ligands in cells and tissues, and nucleic acid hybridization. In a previous study, we demonstrated the applicability of ELF-97 phosphate for detecting endogenous AP activity by flow cytometry. In this study, we show that the spectral characteristics and high signal-to-noise ratio provided by the ELF-97 phosphate make it a useful label for immunodetection via flow cytometry. It can be combined with a variety of other fluorochromes for multiparametric flow cytometry analysis of both endogenous AP activity and intracellular and extracellular immunolabeling with AP-conjugated antibodies. METHODS: ELF-97 phosphate detection of endogenous AP activity in UMR-106 rat osteosarcoma cells was combined with intracellular antigen detection using Oregon Green 488 dye-conjugated secondary antibodies and DNA content analysis using propidium iodide (PI) or 7-aminoactinomycin D (7-AAD). ELF-97 phosphate detection of endogenous AP was also tested for spectral compatibility with a variety of other commonly used fluorochromes. ELF-97 phosphate was then used to directly label intracellular antigens via AP-conjugated antibodies, again combined with the analysis of DNA content using PI and 7-AAD. ELF-97 phosphate was also used to directly detect extracellular antigens. It was combined with Oregon Green 488 dye, phycoerythrin (PE), and PE-Cy5 dye-labeled antibodies for simultaneous four-color analysis. All samples were analyzed on a dual-beam flow cytometer, with UV excitation of the ELF-97 alcohol reaction product. RESULTS: Application of the ELF-97 phosphate to detect AP was found to be compatible with immunodetection and DNA staining techniques. It was also spectrally compatible with a variety of other fluorochromes. Endogenous AP activity could be detected simultaneously with both intracellular antigens labeled using Oregon Green 488 dye, PE, Cy5 dye and Alexa Fluor 568 dye-conjugated antibodies, and DNA content analysis with PI or 7-AAD. This multiparametric assay accurately delineated the distribution of AP in cycling cells and was able to identify cell subsets with varying endogenous AP levels. The ELF-97 alcohol reaction product was found to be an effective label for intracellular antigen immunolabeling with AP-conjugated reagents, and could also be combined with PI and 7-AAD. ELF-97 phosphate was also found to be a useful label for extracellular antigen immunolabeling with AP conjugates, and was compatible with Oregon Green 488 dye, PE, and PE-Cy5 dye-labeled antibodies for four-color surface labeling with minimal spectral overlap and color compensation. CONCLUSIONS: ELF-97 phosphate was shown to be a useful label for both endogenous and antibody-conjugated AP activity as detected by flow cytometry. Its spectral characteristics allow it to be combined with a variety of fluorochromes for multiparametric analysis. Cytometry 43:117-125, 2001. Published 2001 Wiley-Liss, Inc.     

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

Ectoenzymes in Prorocentrum minimum

Ectoenzymes, or enzymes associated with the cell-surface or periplasmic space, play an important role in organic matter cycling by rendering certain forms of dissolved organic matter bioavailable. Ectoenzyme activities may thereby help meet the nutritional demands of harmful algae such as Prorocentrum minimum. The activities of two ectoenzymes; leucine aminopeptidase and alkaline phosphatase, have been studied in axenic cultures of P. minimum. Leucine aminopeptidase releases non-polar amino acids such as leucine from the N-terminus of polypeptides, whereas alkaline phosphatase is an enzyme that is able to hydrolyze phosphate from phosphomonoesters. P. minimum alkaline phosphatase is the better studied of the two ectoenzymes and its characteristics are reviewed herein. Future research on P. minimum physiology will benefit from a growing suite of tools available for assessing the activity of alkaline phosphatase and other ectoenzymes in field populations and ultimately the work done with P. minimum will be useful for studies of other harmful species.     

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.     

A SINGLE-CELL IMMUNOASSAY FOR PHOSPHATE STRESS IN THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)

Current techniques for studying phytoplankton physiology in the field, such as measurements of biochemical activities, nutrient addition bioassays, and determination of photosynthetic efficiency, are useful for assessing the physiology of the bulk community but suffer from a lack of specificity. This would be improved by the development of single-cell methods for monitoring in situ physiology. Here we develop and test an antibody-based assay for identifying phosphate stress in the model dinoflagellate Prorocentrum minimum (Pavillard) Schiller. Antiserum was raised against a cell-surface alkaline phosphatase purified from P. minimum. Western screening indicated that the antiserum reacted with phosphate-stressed cells but not nitrate-stressed or phosphate-replete cells in culture. Immunodepletion confirmed the identification of this protein as an alkaline phosphatase. Based on Western blots, the antiserum appeared to be specific for phosphate-regulated proteins in P. minimum because there is no discernible cross-reaction with closely related P. micans. A whole-cell immunofluorescence assay was used to identify phosphate stress in field populations of P. minimum from Narragansett Bay, Rhode Island. The percentage of labeled P. minimum cells in this environment during the summer of 1998 decreased through time as the inorganic phosphate concentration increased. The percentage of antibody-labeled cells significantly correlated with the percentage of ELF-97-labeled cells determined as another single-cell assay of phosphate stress. This is the first antibody-based method developed for monitoring cell-specific physiology in a dinoflagellate, and the method described here may serve as a model for developing similar tools in other species of phytoplankton.     

SPECIES‐SPECIFIC RESPONSE IN ALKALINE PHOSPHATASE ACTIVITY OF FRESHWATER PHYTOPLANKTON IN A NUTRIENT ENRICHMENT EXPERIMENT

A new method was utilized to study species‐specific responses of phytoplankton to phosphorus limitation in a nutrient enrichment experiment. A substrate, ELF, produces a fluorescent precipitate at the sites of alkaline phosphatase (AP), which makes it possible to visually detect phosphorus (P) limitation in individual cells of multiple species. Lake water was incubated in the laboratory to induce nitrogen (N) or P limitation. Initially, little or no ELF labeling was observed for any of the phytoplankton species, indicating a general lack of P limitation. This observation was supported by low bulk AP activity in the initial field samples. During the experiment, several chlorophyte taxa (Coelastrum, Eudorina, a solitary spiny coccoid) were driven to P limitation, as evidenced by a high percentage of cells displaying ELF labeling when inorganic N was added. Taxa such as Actinastrum and Dictyosphaerium, on the contrary, were never P limited. Little or no ELF was observed in cyanobacterial species, suggesting that P limitation was not achieved in these organisms. Using traditional bulk AP activity, significantly higher levels of AP activity were observed in treatments with inorganic N additions, compared to those with phosphate additions. ELF labeling generally followed the trend of bulk AP, except in species that did not dominate the biomass. Finally, we noted that all species observed were ELF labeled at least on one occasion, except for fragile flagellates which did not withstand the labeling procedure.     

Molecular mechanism of glucose-6-phosphate utilization in the dinoflagellate Karenia mikimotoi

Phosphorus (P) is an essential nutrient for marine phytoplankton as for other living organisms, and the preferred form, dissolved inorganic phosphate (DIP), is often quickly depleted in the sunlit layer of the ocean. Phytoplankton have developed mechanisms to utilize organic forms of P (DOP). Hydrolysis of DOP to release DIP by alkaline phosphatase is believed to be the most common mechanism of DOP utilization. Little effort has been made, however, to understand other potential molecular mechanisms of utilizing different types of DOP. This study investigated the bioavailability of glucose-6-phosphate (G6P) and its underlying molecular mechanism in the dinoflagellate Karenia mikimotoi. Suppression Subtraction Hybridization (SSH) was used to identify genes up- and down-regulated during G6P utilization compared to DIP condition. The results showed that G6P supported the growth and yield of K. mikimotoi as efficiently as DIP. Neither DIP release nor AP activity was detected in the cultures grown in G6P medium, however, suggesting direct uptake of G6P. SSH analysis and RT-qPCR results showed evidence of metabolic modifications, particularly that mitochondrial ATP synthase f1 gamma subunit and thioredoxin reductase were up-regulated while diphosphatase and pyrophosphatase were down-regulated in the G6P cultures. All the results indicate that K. mikimotoi has developed a mechanism other than alkaline phosphatase to utilize G6P.     

RANGE EXTENSION OF CUTLERIA HANCOCKII (CUTLERIALES; PHAEOPHYTA) TO THE SOUTHWESTERN GULF OF CALIFORNIA, MÉXICO

A new method was utilized to study species-specific responses of phytoplankton to phosphorus limitation in a nutrient enrichment experiment. A substrate, ELF, produces a fluorescent precipitate at the sites of alkaline phosphatase (AP), which makes it possible to visually detect phosphorus (P) limitation in individual cells of multiple species. Lake water was incubated in the laboratory to induce nitrogen (N) or P limitation. Initially, little or no ELF labeling was observed for any of the phytoplankton species, indicating a general lack of P limitation. This observation was supported by low bulk AP activity in the initial field samples. During the experiment, several chlorophyte taxa ( Coelastrum , Eudorina , a solitary spiny coccoid) were driven to P limitation, as evidenced by a high percentage of cells displaying ELF labeling when inorganic N was added. Taxa such as Actinastrum and Dictyosphaerium , on the contrary, were never P limited. Little or no ELF was observed in cyanobacterial species, suggesting that P limitation was not achieved in these organisms. Using traditional bulk AP activity, significantly higher levels of AP activity were observed in treatments with inorganic N additions, compared to those with phosphate additions. ELF labeling generally followed the trend of bulk AP, except in species that did not dominate the biomass. Finally, we noted that all species observed were ELF labeled at least on one occasion, except for fragile flagellates which did not withstand the labeling procedure.     

Alkaline and acid phosphatases of the marine diatoms Chaetoceros affinis var. willei (Gran) Hustedt and Skeletonema costatum (Grev.) Cleve

Alkaline phosphatase activity in cultures of the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt was higher than in Skeletonema costatum (Grev.) Cleve. The enzyme activity was localized in coarse cell particles. Acid phosphatase activity was found in the cytoplasmic fraction. Induction of alkaline phosphatase depended on the $\text{N}\text{P}$ ratio in the culture medium. A $\text{N}\text{P}$ ratio > 40 in dilution/batch culture and > 30 in large scale batch culture, respectively, induced alkaline phosphatase.Cell phosphorus showed a critical value below which alkaline phosphatase was induced. Alkaline phosphatase in natural phytoplankton from the Trondheimsfjord is unlikely to occur except possibly in special situations.     

Allelopathic effects of the dinophyte Prorocentrum minimum on the growth of the bacillariophyte Skeletonema costatum

We investigated growth interactions between the dinophyte Prorocentrum minimum and the bacillariophyte Skeletonema costatum using bi-algal cultures under axenic conditions. When low cell densities of P. minimum and high cell densities of S. costatum were inoculated into the same medium, growth of P. minimum was suppressed. Other inoculum combinations resulted in reduced S. costatum maximum cell densities. A mathematical model was used to simulate growth and interactions of P. minimum and S. costatum in bi-algal cultures. The model indicated that P. minimum always outcompeted S. costatum over time. Enriched filtrate from low-density P. minimum cultures significantly stimulated S. costatum growth, but enriched filtrate from high-density P. minimum cultures notably inhibited the growth of S. costatum. Growth of P. minimum was not affected by enriched filtrate from cultures of P. minimum at any density. Filtrates of P. minimum cultures were fractionated by ultrafiltration (molecular weight cutoff >3000 Da), and retentate that included polysaccharide(s) significantly inhibited the growth of S. costatum.     

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.     

Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: Activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation

Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified dolichol phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in V_max with no alteration in the apparent K_m for GDP-Manose. Phosphorylation with [γ-³²P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation–dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.     

Characterization of a bone-specific alkaline phosphatase in chick limb mesenchymal cell cultures

Previous morphometric and biochemical studies suggested that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. Others have shown that osteoblast expression is marked by an increase in bone-specific alkaline phosphatase activity. Our results indicate that chick limb mesenchymal cells develop alkaline phosphatase activity that is identical to that of the chick embryonic bone-specific isoenzyme. The alkaline phosphatase isozymes were partially purified from samples of chick intestine, liver, stage 38 embryonic limbs, and cultures of stage 24 limb mesenchymal cells. These tissues were separately extracted with butanol, acetone precipitated, redissolved, and passed over a DEAE-Sephacel ion-exchange column and ion-filtration column (Sephadex A-25). From the data obtained during this purification scheme, we conclude that the alkaline phosphatase from stage 38 limbs (bones) and Day 4 cultures are identical, and this activity is different from the enzyme purified from intestine and liver. The cell culture isozyme has an apparent K_m, heat lability, response to specific inhibitors, electrophoretic mobility, and molecular weight similar to those of bone-specific alkaline phosphatase. These observations support the view that osteoblastic progenitor cells are present in the stage 24 limb mesenchyme and that under specific culture conditions, bone development can be uniquely observed in vitro.     

Alkaline phosphatase activity in whitefly salivary glands and saliva

Alkaline phosphatase activity was histochemically localized in adult whiteflies (Bemisia tabaci B biotype, syn. B. argentifolii) with a chromogenic substrate (5-bromo-4-chloro-3-indolylphosphate) and a fluorogenic substrate (ELF-97). The greatest amount of staining was in the basal regions of adult salivary glands with additional activity traced into the connecting salivary ducts. Other tissues that had alkaline phosphatase activity were the accessory salivary glands, the midgut, the portion of the ovariole surrounding the terminal oocyte, and the colleterial gland. Whitefly nymphs had activity in salivary ducts, whereas activity was not detected in two aphid species (Rhodobium porosum and Aphis gossypii). Whitefly diet (15% sucrose) was collected from whitefly feeding chambers and found to have alkaline phosphatase activity, indicating the enzyme was secreted in saliva. Further studies with salivary alkaline phosphatase collected from diet indicated that the enzyme had a pH optimum of 10.4 and was inhibited by 1 mM cysteine and to a lesser extent 1 mM histidine. Dithiothreitol, inorganic phosphate, and ethylenediaminetetraacetic acid (EDTA) also inhibited activity, whereas levamisole only partially inhibited salivary alkaline phosphatase. The enzyme was heat tolerant and retained approximately 50% activity after a 1-h treatment at 65 degrees C. The amount of alkaline phosphatase activity secreted by whiteflies increased under conditions that stimulate increased feeding. These observations indicate alkaline phosphatase may play a role during whitefly feeding.     

Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2

We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M_r 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M_r 160,000 inhibit phosphatase activity by approximately 70%.     

Fluorometric measurement of alkaline phosphatase activity in algae

SUMMARY. Using both cultures of algae and natural populations, alkaline phosphatase activity located on the cell surface has been measured by a fluorometric procedure. This was done in order to establish optimum standard conditions for the measurement of this activity as an indicator of phosphorus deficiency and to provide a means of interpreting alkaline phosphatase measurements on natural phytoplankton populations. A concentration of 10 μM o-methylfluorescein phosphate saturates or nearly saturates the reaction in a variety of situations. In most trials, rates increased with temperature to or beyond 35°C. Optimum pH values in the range 7–10 were observed. In six of the algae examined, maximum alkaline phosphatase activities were dependent on external calcium at 100 μM or more. One alga, Synura uvella , showed acid phosphatase activity, peaking at pH 5–6, induced by phosphorus deficiency. Based on comparisons between P-sufficient and P-deficient cultures, alkaline phosphatase activities in excess of 0.1 μmol o-methylfluorescein phosphate hydrolysed per mg dry weight per h or 0.1 μmol per μg ATP per h are suggested as indicative of phosphorus deficiency.     

Phosphorus strategy in bloom-forming cyanobacteria (Dolichospermum and Microcystis) and its role in their succession

Dolichospermum (formerly Anabaena) and Microcystis cause harmful cyanobacterial blooms in freshwater ecosystems worldwide. Input reduction of both nitrogen (N) and phosphorus (P) are commonly recognized as basic ways of controlling blooms, but little is known about the roles of nutrients and their using strategy among cyanobacteria in triggering the succession of diazotrophic to non-diazotrophic cyanobacteria. In this study, we investigated in situ responses of cyanobactria to ambient P status during the transition from Dolichospermum flos-aquae to Microcystis spp. in Lake Taihu and Lake Chaohu. While dominant in phytoplankton community, D. flos-aquae experienced P deficiency as evidenced by qualitative detection of extracellular phosphatase via enzyme labeled fluorescence (ELF). The percentage of ELF-labelled D. flos-aquae cells was 33% when it dominated the phytoplankton community, and was 78% when it co-dominated with Microcystis spp., indicating an increase in P deficiency. Meanwhile, no ELF-labelled Microcystis cells were observed while polyphosphate body (PPB) were present, suggesting that Microcystis spp. were not P deficient. Additionally, the percentages of Microcystis cells containing PPB showed an inverted “U-shaped” relationship with concentrations on soluble reactive phosphorus (SRP). To validate the field observation, a laboratory study of the monocultures of the dominant cyanobacteria was conducted. Extracellular alkaline phosphatase activity (APA) and PPB accumulation were regulated by P availability in monocultures of D. flos-aquae. Interestingly, no cell bound extracellular phosphatase was found on Microcystis aeruginasa even in the culture without P supply. Consistently, the expressions of phosphatase encoding gene phoX showed no differences among the treatments. The way in which PPB accumulation occurred in Microcystis spp. in response to P availability in the cultures was similar to that observed in the field, demonstrating a strategy of energy conservation over P accumulation. The competitive advantage of Microcystis spp. was displayed at low P concentrations: where it could rapidly uptake and store inorganic P, which also increased the P deficiency of the coexisting phytoplankton species. Responses of P-transport gene pstS confirmed this hypothesis. The physiological and molecular mechanisms mentioned above enable Microcystis to survive and proliferate in environment with low available P supply more efficiently. In conclusion, different cyanobacterial species have distinct ways of responding to P availability, suggesting that the control of cyanobacterial blooms by targeted nutrient reduction is largely dependent upon the dominant species. P reduction is more effective in controlling diazotrophic cyanobacteria than non-diazotrophic cyanobacteria.