Effect of polyphosphate metabolism on the Escherichia coli phosphate-starvation response.

A previously developed dynamic model of the Escherichia coli Pho regulon was extended to investigate the effect of polyphosphate synthesis and degradation on this control system. Differential equations for ATP and polyphosphate were formulated, and the model was applied to the growth of cells containing the ppk and ppx genes under control of separate, inducible promoters. In agreement with recent experimental observations, the degradation of polyphosphate by PPX during a period of phosphate limitation could repress the phosphate-starvation response. This is attributed to the release of phosphate from the cell into the periplasm, where it can be detected by the external phosphate sensor. A segregated model was then developed to account for differences in K(I), the dissociation constant for the repression complex, among cells of the population. Since K(I) is the key parameter in determining whether the Pho response is induced or repressed at a particular surface phosphate concentration, this permitted the induction of some cells while others remained repressed. The induction profiles resulting from the population-averaged values more closely matched experimental results than did those with the nonsegregated model.     

Engineering polyphosphate metabolism in Escherichia coli: implications for bioremediation of inorganic contaminants

Polyphosphate metabolism plays an important role in the bioremediation of phosphate contamination in municipal wastewater, and may play a key role in heavy metal tolerance and bioremediation. However, little is known about the regulation of polyphosphate metabolism in microorganisms and its role in heavy metal toxicity. We have manipulated polyphosphate metabolism in Escherichia coli by overexpressing the genes for polyphosphate kinase (ppk) and for polyphosphatase (ppx) under control of their native promoters and inducible promoters. Overexpression of ppk results in high levels of intracellular polyphosphate, improved phosphate uptake, but no increase in tolerance to heavy metals. Overexpression of both ppk and ppx results in lower levels of intracellular polyphosphate, secretion of phosphate from the cell, and increased tolerance to heavy metals. Metabolic flux analysis indicates that the cell responds to increased flux through the PPK-PPX pathway by altering flux through the TCA cycle.     

Genetic manipulation of polyphosphate metabolism affects cadmium tolerance in Escherichia coli.

The polyphosphate metabolic pathways in Escherichia coli were genetically manipulated to test the effect of polyphosphate on tolerance to cadmium. A polyphosphate kinase (ppk) and polyphosphatase (ppx) mutant strain produced no polyphosphate, whereas the same strain carrying multiple copies of ppk on a high-copy plasmid produced significant quantities. The doubling times of both strains increased with increasing cadmium concentrations. In contrast, the mutant strain carrying multiple copies of ppk and ppx produced 1/20 of the polyphosphate found in the strain carrying multiple copies of ppk only and showed no significant increase in doubling time over the same cadmium concentration range.     

Manipulation of independent synthesis and degradation of polyphosphate in Escherichia coli for investigation of phosphate secretion from the cell.

The genes involved in polyphosphate metabolism in Escherichia coli were cloned behind different inducible promoters on separate plasmids. The gene coding for polyphosphate kinase (PPK), the enzyme responsible for polyphosphate synthesis, was placed behind the Ptac promoter. Polyphosphatase, a polyphosphate depolymerase, was similarly expressed by using the arabinose-inducible PBAD promoter. The ability of cells containing these constructs to produce active enzymes only when induced was confirmed by polyphosphate extraction, enzyme assays, and RNA analysis. The inducer concentrations giving optimal expression of each enzyme were determined. Experiments were performed in which ppk was induced early in growth, overproducing PPK and allowing large amounts of polyphosphate to accumulate (80 mumol in phosphate monomer units per g of dry cell weight). The ppx gene was subsequently induced, and polyphosphate was degraded to inorganic phosphate. Approximately half of this polyphosphate was depleted in 210 min. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells and was secreted into the medium, leading to a down-regulation of the phosphate-starvation response. In addition, the steady-state polyphosphate level was precisely controlled by manipulating the degree of ppx induction. The polyphosphate content varied from 98 to 12 mumol in phosphate monomer units per g of dry cell weight as the arabinose concentration was increased from 0 to 0.02% by weight.     

Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830

Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P. aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of P. aeruginosa 8830. Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that ppk is transcribed from a sigmaE-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.     

The gene for an exopolyphosphatase of Pseudomonas aeruginosa.

In Pseudomonas aeriginosa, a gene, ppx, that encodes exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] of 506 amino acids (56,419 Da) was found downstream of the gene for polyphosphate kinase, ppk. Since ppx is located in the opposite direction of the ppk gene, they do not constitute an operon. The predicted amino acid sequence of PPX is 41% identical with Escherichia coli PPX. The gene product of ppx (paPPX) was overproduced in E. coli, and its activity was evaluated. Orthophosphate (Pi) is released from polyphosphate [poly(P)], the average chain lengths of which are 79 and 750, respectively. The amount of Pi released matched the amount of poly(P) lost. Thus ppx encodes an enzyme that has exopoly(P)ase activity.     

Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.     

Genetic improvement of Escherichia coli for enhanced biological removal of phosphate from wastewater.

The ability of Escherichia coli MV1184 to accumulate inorganic phosphate (Pi) was enhanced by manipulating the genes involved in the transport and metabolism of Pi. The high-level Pi accumulation was achieved by modifying the genetic regulation and increasing the dosage of the E. coli genes encoding polyphosphate kinase (ppk), acetate kinase (ackA), and the phosphate-inducible transport system (pstS, pstC, pstA, and pstB). Acetate kinase was employed as an ATP regeneration system for polyphosphate synthesis. Recombinant strains, which contained either pBC29 (carrying ppk) or pEP02.2 (pst operon), removed approximately two- and threefold, respectively, more Pi from minimal medium than did the control strain. The highest rates of Pii removal were obtained by strain MV1184 containing pEP03 (ppk and ackA). However, unlike the control strain, MV1184 (pEP03) released Pi to the medium after growth had stopped. Drastic changes in growth and Pi uptake were observed when pBC29 (ppk) and pEP02.2 (pst operon) were introduced simultaneously into MV1184. Even though growth of this recombinant was severely limited in minimal medium, the recombinant could remove approximately threefold more Pi than the control strain. Consequently, the phosphorus content of this recombinant reached a maximum of approximately 16% on a dry weight basis (49% as phosphate).     

脑膜炎大肠杆菌K1株ppk1基因致病机制初探

【目的】构建脑膜炎大肠杆菌K1(Escherichia coli,E.coli K1)株E44的聚磷酸盐激酶1(Polyphosphate kinase 1,PPK1)基因敲除株,并对其生物学功能进行初步研究,为明确ppk1基因在E.coli K1株致脑膜炎机制中的作用奠定基础。【方法】利用自杀质粒pCVD442及基因同源重组技术敲除E.coli K1株E44中的ppk1基因,构建ppk1缺失突变株Δppk1;体外比较野生株和突变株在低营养及氧化压力情况下的生存能力;考察二者对人脑微血管内皮细胞(Human brain microvascular endothelial cells,HBMEC)的黏附能力;通过测定乳酸脱氢酶(Lactic dehydrogenase,LDH)释放活性,比较野生株和突变株对HBMEC的损伤效应。【结果】PCR及序列分析证实,突变株缺失全长ppk1基因。与野生株E44相比,ppk1突变株Δppk1在低营养环境中和氧化刺激条件下的生存能力明显降低。相对于E44,Δppk1对HBMEC的黏附能力减弱。与HBMEC孵育后,突变株孵育组HBMEC的LDH释放活性明显低于野生株孵育组。【结论】ppk1对E.coli K1株E44在低营养环境中的生存、抵抗氧化压力,以及黏附HBMEC和对细胞的毒性损伤有重要作用。     

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner

Filamentous microorganisms of the bacterial genus Streptomyces have a complex life cycle that includes physiological and morphological differentiations. It is now fairly well accepted that lysis of Streptomyces vegetative mycelium induced by programmed cell death (PCD) provides the required nutritive sources for the bacterium to erect spore-forming aerial hyphae. However, little is known regarding cellular compounds released during PCD and the contribution of these molecules to the feeding of surviving cells in order to allow them to reach the late stages of the developmental program. In this work we assessed the effect of extracellular sugar phosphates (that are likely to be released in the environment upon cell lysis) on the differentiation processes. We demonstrated that the supply of phosphorylated sugars, under inorganic phosphate limitation, delays the occurrence of the second round of PCD, blocks streptomycetes life cycle at the vegetative state and inhibits antibiotic production. The mechanism by which sugar phosphates affect development was shown to involve genes of the Pho regulon that are under the positive control of the two component system PhoR/PhoP. Indeed, the inactivation of the response regulator phoP of Streptomyces lividans prevented the 'sugar phosphate effect' whereas the S. lividans ppk (polyphosphate kinase) deletion mutant, known to overexpress the Pho regulon, presented an enhanced response to phosphorylated sugars.     

Phosphite disrupts the acclimation of Saccharomyces cerevisiae to phosphate starvation.

The influence of phosphite (H2PO3-) on the response of Saccharomyces cerevisiae to orthophosphate (HPO4(2-); Pi) starvation was assessed. Phosphate-repressible acid phosphatase (rAPase) derepression and cell development were abolished when phosphate-sufficient (+Pi) yeast were subcultured into phosphate-deficient (-Pi) media containing 0.1 mM phosphite. By contrast, treatment with 0.1 mM phosphite exerted no influence on rAPase activity or growth of +Pi cells. 31P NMR spectroscopy revealed that phosphite is assimilated and concentrated by yeast cultured with 0.1 mM phosphite, and that the levels of sugar phosphates, pyrophosphate, and particularly polyphosphate were significantly reduced in the phosphite-treated -Pi cells. Examination of phosphite's effects on two PHO regulon mutants that constitutively express rAPase indicated that (i) a potential target for phosphite's action in -Pi yeast is Pho84 (plasmalemma high-affinity Pi transporter and component of a putative phosphate sensor-complex), and that (ii) an additional mechanism exists to control rAPase expression that is independent of Pho85 (cyclin-dependent protein kinase). Marked accumulation of polyphosphate in the delta pho85 mutant suggested that Pho85 contributes to the control of polyphosphate metabolism. Results are consistent with the hypothesis that phosphite obstructs the signaling pathway by which S. cerevisiae perceives and responds to phosphate deprivation at the molecular level.     

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins.

We observed that wild-type Escherichia coli utilized a linear polyphosphate with a chain length of 100 phosphate residues (poly-P100) as the sole source of phosphate in growth medium. A mutation in the gene phoA of alkaline phosphatase or phoB, the positive regulatory gene, prevented growth in this medium. Since no alkaline phosphatase activity was detected outside the wild-type cells, the periplasmic presence of the enzyme was necessary for the degradation of polyphosphate. A 90% reduction in the activity of periplasmic acid phosphatase with a pH optimum of 2.5 (delta appA mutants) did not affect polyphosphate utilization. Of the porins analyzed (OmpC, OmpF, and PhoE), the phoB-inducible porin PhoE was not essential since its absence did not prevent growth. To study how poly-P100 diffused into the cells, we used high-resolution 31P nuclear magnetic resonance (31P NMR) spectroscopy. The results suggest that poly-P100 entered the periplasm and remained in equilibrium between the periplasm and the medium. When present individually, porins PhoE and OmpF facilitated a higher permeability for poly-P100 than porin OmpC did. The degradation of polyphosphate by intact cells of E. coli observed by 31P NMR showed a time-dependent increase in cellular phosphate and a decrease in polyphosphate concentration.     

Bacterial microcompartment‐directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli

Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate‐synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co‐expression of E. coli PPK1 fused with a microcompartment‐targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8‐fold compared with non‐targeted PPK1. Co‐expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co‐expression of PPX with non‐targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal.     

Polyphosphate stores enhance the ability of Vibrio cholerae to overcome environmental stresses in a low-phosphate environment

Vibrio cholerae, the causative agent of Asiatic cholera, has been reported to make large quantities of polyphosphate. Inorganic polyphosphate is a ubiquitous molecule with a variety of functions in prokaryotic and eukaryotic cells. We constructed a V. cholerae mutant with a deletion in the polyphosphate kinase (ppk) gene. The mutant was defective in polyphosphate biosynthesis. Deletion of ppk had no significant effect on production of cholera toxin, hemagglutinin/protease, motility, biofilm formation, and colonization of the suckling mouse intestine. The wild type and mutant had similar growth rates in rich and minimal medium and exhibited similar phosphate uptake and alkaline phosphatase induction. In contrast to ppk mutants from other gram-negative bacteria, the V. cholerae mutant survived prolonged starvation in LB medium and artificial seawater basal salts. The ppk mutant was significantly more sensitive to low pH, high salinity, and oxidative stress when it was cultured in low-phosphate minimal medium. The ppk mutant failed to induce catalase when it was downshifted to phosphorus-limiting conditions. Furthermore, the increased sensitivity of the ppk mutant to environmental stressors in phosphate-limited medium correlated with a diminished capacity to synthesize ATP from intracellular reservoirs. We concluded that polyphosphate protects V. cholerae from environmental stresses under phosphate limitation conditions. It has been proposed that toxigenic V. cholerae can survive in estuaries and brackish waters in which phosphorus and/or nitrogen can be a limiting nutrient. Thus, synthesis of large polyphosphate stores could enhance the ability of V. cholerae to survive in the aquatic environment.     

Regulation of cation-coupled high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Studies of the high-affinity phosphate transporters in the yeast Saccharomyces cerevisiae using mutant strains lacking either the Pho84 or the Pho89 permease revealed that the transporters are differentially regulated. Although both genes are induced by phosphate starvation, activation of the Pho89 transporter precedes that of the Pho84 transporter early in the growth phase in a way which may possibly reflect a fine tuning of the phosphate uptake process relative to the availability of external phosphate.     

Low affinity orthophosphate carriers regulate PHO gene expression independently of internal orthophosphate concentration in Saccharomyces cerevisiae

Phosphate is an essential nutrient that must be taken up from the growth medium through specific transporters. In Saccharomyces cerevisiae, both high and low affinity orthophosphate carriers allow this micro-organism to cope with environmental variations. Intriguingly, in this study we found a tight correlation between selenite resistance and expression of the high affinity orthophosphate carrier Pho84p. Our work further revealed that mutations in the low affinity orthophosphate carrier genes (PHO87, PHO90, and PHO91) cause deregulation of phosphate-repressed genes. Strikingly, the deregulation due to pho87Delta, pho90Delta, or pho91Delta mutations was neither correlated to impaired orthophosphate uptake capacity nor to a decrease of the intracellular orthophosphate or polyphosphate pools, as shown by (31)P NMR spectroscopy. Thus, our data clearly establish that the low affinity orthophosphate carriers affect phosphate regulation independently of intracellular orthophosphate concentration through a new signaling pathway that was found to strictly require the cyclin-dependent kinase inhibitor Pho81p. We propose that phosphate-regulated gene expression is under the control of two different regulatory signals as follows: the sensing of internal orthophosphate by a yet unidentified protein and the sensing of external orthophosphate by low affinity orthophosphate transporters; the former would be required to maintain phosphate homeostasis, and the latter would keep the cell informed on the medium phosphate richness.