Characterization of isolated acidocalcisomes from Toxoplasma gondii tachyzoites reveals a novel pool of hydrolyzable polyphosphate

Toxoplasma gondii tachyzoites were fractionated by modification of an iodixanol density gradient method previously used for acidocalcisome isolation from Trypanosoma cruzi epimastigotes. Fractions were characterized using electron microscopy, x-ray microanalysis, and enzymatic markers, and it was demonstrated that the heaviest (pellet) fraction contains electron-dense vacuoles rich in phosphorus, calcium, and magnesium, as found before for acidocalcisomes. Staining with 4',6-diamidino-2-phenylindole (DAPI) indicated that poly- phosphate (polyP) was preferentially localized in this fraction together with pyrophosphate (PP(i)). Using an enzyme-based method, millimolar levels (in terms of P(i) residues) of polyP chains of less than 50 residues long and micromolar levels in polyP chains of about 700-800 residues long were found to be preferentially localized in this fraction. The fraction also contained the pyrophosphatase and polyphosphatase activities characteristic of acidocalcisomes. Western blot analysis using antibodies against proteins from micronemes, dense granules, rhoptries, and plasma membrane showed that the acidocalcisomal fraction was not contaminated by these other organelles. T. gondii polyP levels rapidly decreased upon exposure of the parasites to a calcium ionophore (ionomycin), to an inhibitor of the V-H(+)-ATPase (bafilomycin A(1)), or to the alkalinizing agent NH(4)Cl. These changes were in parallel to an increase in intracellular Ca(2+) concentration, suggesting a close association between polyP hydrolysis and Ca(2+) release from the acidocalcisome. These results provide a useful method for the isolation and characterization of acidocalcisomes, showing that they are distinct from other previously recognized organelles present in T. gondii, and provide evidence for the role of polyP metabolism in response to cellular stress.     

The Acidocalcisome Vacuolar Transporter Chaperone 4 Catalyzes the Synthesis of Polyphosphate in Insect‐stages of Trypanosoma brucei and T. cruzi

Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium‐rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno‐electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites.     

Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.     

The H(+)-pyrophosphatase of Rhodospirillum rubrum is predominantly located in polyphosphate-rich acidocalcisomes

Acidocalcisomes are acidic, calcium storage compartments with a H(+) pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H(+)-pyrophosphatase (H(+)-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H(+)-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H(+)-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection

Polyphosphate (polyP) is an anionic polymer of orthophosphate groups linked by high energy bonds that typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes. PolyP synthesis in eukaryotes was unclear until it was demonstrated that the protein named Vtc4p (vacuolar transporter chaperone 4) is a long chain polyP kinase that localizes to the yeast vacuole. Here, we report that TbVtc4 (Vtc4 ortholog of Trypanosoma brucei) encodes, in contrast, a short chain polyP kinase that localizes to acidocalcisomes. The subcellular localization of TbVtc4 was demonstrated by fluorescence and electron microscopy of cell lines expressing TbVtc4 in its endogenous locus fused to an epitope tag and by purified polyclonal antibodies against TbVtc4. Recombinant TbVtc4 was expressed in bacteria, and polyP kinase activity was assayed in vitro. The in vitro growth of conditional knock-out bloodstream form trypanosomes (TbVtc4-KO) was significantly affected relative to the parental cell line. This mutant had reduced polyP kinase activity and short chain polyP content and was considerably less virulent in mice. The wild-type phenotype was recovered when an ectopic copy of the TbVtc4 gene was expressed in the presence of doxycycline. The mutant also exhibited a defect in volume recovery under osmotic stress conditions in vitro, underscoring the relevance of polyP in osmoregulation.     

The acidocalcisome Ca2+-ATPase (TgA1) of Toxoplasma gondii is required for polyphosphate storage, intracellular calcium homeostasis and virulence

A large proportion of intracellular Ca2+ in Toxoplasma gondii tachyzoites is stored within acidocalcisomes. These organelles are characterized by their acidic nature and high calcium and polyphosphate (polyP) content. The activity of a Ca2+/H+-ATPase named TgA1 may be important for the accumulation of Ca2+ in these organelles. This enzyme belongs to a group of plasma membrane Ca2+-ATPase (PMCA) that lack a calmodulin-binding domain and have vacuolar localization. To investigate the role of this enzyme, we have generated T. gondii mutants deficient in TgA1 through gene disruption. Proliferation of these mutants decreased dramatically because of deficient cell invasion. In addition, these cells had reduced virulence in a mouse model. Biochemical analysis revealed that the cell polyP content was drastically reduced, and the basal calcium levels were increased and unstable. Microneme secretion under the conditions of stimulation by ionophores was altered. Complementation of null mutants with TgA1 restored most functions. In summary, these results establish a link between TgA1, calcium homeostasis, polyP storage and virulence.     

Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs

High field (31)P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P(2)O(7)(4-)) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 +/- 25.9 and 37.6 +/- 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 +/- 17.0 and 36.0 +/- 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with (32)P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by (31)P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.     

31P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Evidence for high levels of condensed inorganic phosphates

High resolution (31)P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a (1)H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis. Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds. The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate. (31)P NMR spectra of purified T. brucei, T. cruzi, and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates. In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.     

A pyrophosphatase regulating polyphosphate metabolism in acidocalcisomes is essential for Trypanosoma brucei virulence in mice

We report the functional characterization of a soluble pyrophosphatase (TbVSP1), which localizes to acidocalcisomes, a vesicular acidic compartment of Trypanosoma brucei. Depending on the pH and the cofactors Mg(2+) or Zn(2+), both present in the compartment, the enzyme hydrolyzes either inorganic pyrophosphate (PP(i)) (k(cat) = 385 s(-1)) or tripolyP (polyP(3)) and polyphosphate (polyP) of 28 residues (polyP(28)) with k(cat) values of 52 and 3.5 s(-1), respectively. An unusual N-terminal domain of 160 amino acids, containing a putative calcium EF-hand-binding domain, is involved in protein oligomerization. Using double-stranded RNA interference methodology, we produced an inducible bloodstream form (BF) deficient in the TbVSP1 protein (BFiVSP1). The long-chain polyP levels of these mutants were reduced by 60%. Their phenotypes revealed a deficient polyP metabolism, as indicated by their defective response to phosphate starvation and hyposmotic stress. BFiVSP1 did not cause acute virulent infection in mice, demonstrating that TbVSP1 is essential for growth of bloodstream forms in the mammalian host.     

Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism

Polyphosphates (polyPs) have been found in all cell types examined to date and play diverse roles, depending on the cell type. In eukaryotic organisms, polyPs have been mainly investigated in mammalian cells, with few studies on insects. In this study, we investigated mitochondrial polyphosphate metabolism in the red flour beetle, Tribolium castaneum. Substrate specificity for different chain lengths demonstrated the presence of two exopolyphosphatase isoforms in mitochondria. T. castaneum mitochondrial polyP levels decreased after injection with soluble pyrophosphatase (Tc‐sPPase) dsRNA, while the membrane exopolyphosphate activity increased. Mitochondrial respiration modulated exopolyphosphatase activity only in wild‐type beetles. Tripolyphosphate was able to increase the F‐ATPase activity in wild‐type and Tc‐sPPase RNAi beetles. We suggest that inorganic pyrophosphatase modulates polyphosphate metabolism in mitochondria and affects the link between mitochondrial activity and polyphosphate metabolism in T. castaneum.     

A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi

Acidocalcisomes are dense, acidic organelles with a high concentration of phosphorus present as pyrophosphate and polyphosphate complexed with calcium and other cations. Acidocalcisomes have been linked to the contractile vacuole complex in Chlamydomonas reinhardtii, Dictyostelium discoideum, and Trypanosoma cruzi. A microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex in T. cruzi results in translocation of aquaporin and the resulting water movement which, in addition to swelling of acidocalcisomes, is responsible for the volume reversal not accounted for by efflux of osmolytes. Polyphosphate hydrolysis occurs during hyposmotic stress, probably increasing the osmotic pressure of the contractile vacuole and facilitating water movement.     

Acidocalcisomes as calcium- and polyphosphate-storage compartments during embryogenesis of the insect Rhodnius prolixus Stahl

Background

The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus.

Methodology/Principal findings

Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H⁺-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs.

Conclusions

We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.     

The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.     

Trypanosoma rangeli: Differential expression of cell surface polypeptides and ecto-phosphatase activity in short and long epimastigote forms

Trypanosoma rangeli is a parasite of a numerous wild and domestic animals, presenting wide geographical distribution and high immunological cross-reactivity with Trypanosoma cruzi, which may lead to misdiagnosis. T. rangeli has a complex life cycle, involving distinct morphological and functional forms in the vector. Here, we characterized the cell surface polypeptides and the phosphatase activities in short and long epimastigotes forms of T. rangeli, using intact living parasites. The surface protein profile revealed by the incubation of parasites with biotin showed a preferential expression of the 97, 70, 50, 45, 25-22, and 15 kDa biotinylated polypeptides in the long forms, in contrast to the 55 and 28 kDa biotinylated polypeptides synthesized by the short epimastigotes. Additionally, flow cytometry analysis showed that the short forms had relatively lower biotin surface binding than long ones. The involvement of phosphatases with the trypanosomatid differentiation has been proposed. In this sense, T. rangeli living parasites were able to hydrolyze the artificial substrate p-nitrophenylphosphate at a rate of 25.57+/-2.03 and 10.09+/-0.93 nmol p-NPP x h(-1) x 10(7) cells for the short and long epimastigotes, respectively. These phosphatase activities were linear with time for at least 60 min and the optimum pH lies in the acid range. Classical inhibitors of acid phosphatases, such as ammonium molybdate, sodium fluoride, and zinc chloride, showed a significant decrease in these phosphatase activities, with different patterns of inhibition. Additionally, these phosphatase activities presented different kinetic parameters (Km and Vmax) and distinct sensitivities to divalent cations. Both epimastigotes were unable to release phosphatase to the extracellular environment. Cytochemical analysis demonstrated the localization of these enzymes on the parasite surfaces (cell body and flagellum) and in intracellular vacuoles, resembling acidocalcisomes.     

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2–40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO₃, in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, ³¹P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.     

Stress response to high osmolarity in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.     

An acidocalcisomal exopolyphosphatase from Leishmania major with high affinity for short chain polyphosphate

We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX). The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa. LmPPX differs from other exopolyphosphatases previously investigated. Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg(2+) requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity. LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, or p-nitrophenylphosphate. Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization. High levels of short chain (21.4 +/- 3.0 mm) and long chain polyP (55.9 +/- 5.6 mm) were detected in L. major promastigotes. The unique characteristics of LmPPX and L. major polyP metabolism may facilitate the development of novel antileishmanial agents.     

Polyphosphate kinase regulates error-prone replication by DNA polymerase IV in Escherichia coli

The ppk gene encodes polyphosphate kinase (Ppk), an enzyme that catalyses the polymerization of inorganic phosphate into long chains of polyphosphate (polyP). An insertion mutation in ppk causes a decrease in adaptive mutation in Escherichia coli strain FC40. Adaptive mutation in FC40 mostly results from error-prone DNA polymerase IV (Pol IV), encoded by dinB; most of the antimutagenic phenotype of the ppk mutant disappears in a dinB mutant strain. In addition, the ppk mutant causes a decrease in growth-dependent mutations produced by overexpressing Pol IV. However, the amount of Pol IV protein is unchanged in the ppk mutant strain, indicating that the activity or fidelity of Pol IV is altered. Adaptive mutation is inhibited both by the absence of Ppk, which results in low amounts of polyP, and by overproduction of Ppk, which results in high amounts of polyP, suggesting that an optimal level of polyP is necessary. Taken together, these results suggest a novel mechanism involving polyP that directly or indirectly regulates DNA polymerase activity or fidelity.