Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. Copyright 1998 John Wiley & Sons, Inc.     

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.     

Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 microM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.     

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.     

Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 μM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.     

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.     

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.     

Inorganic polyphosphates and heavy metal resistance in microorganisms

The mechanisms of heavy metal resistance in microbial cells involve multiple pathways. They include the formation of complexes with specific proteins and other compounds, the excretion from the cells via plasma membrane transporters in case of procaryotes, and the compartmentalization of toxic ions in vacuoles, cell wall and other organelles in case of eukaryotes. The relationship between heavy metal tolerance and inorganic polyphosphate metabolism was demonstrated both in prokaryotic and eukaryotic microorganisms. Polyphosphates, being polyanions, are involved in detoxification of heavy metals through complex formation and compartmentalization. The bacteria and fungi cultivated in the presence of some heavy metal cations contain the enhanced levels of polyphosphate. In bacteria, polyphosphate sequesters heavy metals; some of metal cations stimulate an exopolyphosphatase activity, which releases phosphate from polyphosphates, and MeHPO₄^? ions are then transported out of the cells. In fungi, the overcoming of heavy metal stresses is associated with the accumulation of polyphosphates in cytoplasmic inclusions, vacuoles and cell wall and the formation of cation/polyphosphate complexes. The effects of knockout mutations and overexpression of the genes encoding polyphosphate-metabolizing enzymes on heavy metal resistance are discussed.

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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.     

Inorganic polyphosphate interacts with ribosomes and promotes translation fidelity in vitro and in vivo

Inorganic polyphosphate is a biological macromolecule consisting of multiple phosphates linked by high-energy bonds. Polyphosphate occurs in cells from all domains of life, and is known to play roles in a diverse collection of cellular functions. Here we examine the relationship between polyphosphate and protein synthesis in Escherichia coli. We report that polyphosphate associates with E. coli ribosomes in vitro. Characterization of this interaction reveals that both long-chain and short-chain polyphosphates interact with the ribosome. Intact 70S ribosomes, as well as 50S and 30S subunits, display a specific interaction with polyphosphate that is mediated primarily by contacts with ribosomal proteins. Additionally, we examined functional consequences of a ppk mutation, which severely reduces levels of intracellular polyphosphate. Extracts from ppk mutants contain lower levels of polysomes than wild-type cells, suggesting a defect in mRNA utilization or the mRNA-ribosome interaction. Ribosomes from wild-type and ppk mutant cells were isolated, and their activities were compared using a polyU RNA in vitro translation assay. While rates of polyphenylalanine synthesis are similar, use of ribosomes from ppk cells results in a misincorporation rate about five times higher compared with the rate observed when ribosomes from wild-type cells are used. Mistranslation rates in vivo were measured directly, and ppk mutants displayed higher readthrough frequencies for two different stop codons. Taken together, these results indicate that polyphosphate plays an important role in maintaining optimal translation efficiency in vivo and in vitro.     

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.     

Regulation of ppk expression and in vivo function of Ppk in Streptomyces lividans TK24

The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the gamma phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.     

Effectiveness of a microbial formula,as a bioaugmentation agent,tailored for bioremediation of diesel oil and heavy metal co-contaminated soil

The activation of natural bioremediation potentials is the challenge that research is currently addressing for overcoming bottlenecks still affecting bioremediation applications. Bioaugmentation is one possible way to activate such natural potentials, provided that the biodiversity introduced to increase catabolically relevant capacity is identified also considering the ecological context. The present work deals with bioaugmentation aimed at the remediation of a soil co-contaminated (spiked) with both diesel oil (1%, v/w), and heavy metals (Pb and Zn), using intact soil core microcosms in different experimental conditions. We supposed that both heavy metal resistance and active metabolism towards organic pollutants are essential metabolic traits to trap the energetic flux, which drives the microbial community towards biodegradation under the given experimental conditions. Consequently, the bioaugmentation was performed by introducing a tailor made microbial formula composed of 12 allochthonous strains. They belong to a stable population previously isolated from a chronic polluted site and are both hydrocarbon degraders and heavy metal resistant and, also, compatible with the autochthonous microbial community. The active role of the microbial formula in pushing the entire community towards an effective bioremediation of diesel oil close to 75%, in the presence of bioavailable metals, has been proven through hydrocarbons analysis, metabolic and molecular profiling at community level (Biolog system, DGGE).     

AM真菌对重金属污染土壤生物修复的应用与机理

土壤重金属污染威胁人类健康和整个生态系统,而高效、低耗、安全的生物修复技术显示出了极大的应用潜力,特别是利用植物-微生物共生体增强生物修复效应的应用.丛枝菌根(Arbuscular Mycorrhizae,AM)真菌是一类广泛分布于土壤生态系统中的有益微生物,能与90％以上的陆生高等植物形成共生体.研究发现,AM真菌能够增强宿主植物对土壤中重金属胁迫的耐受性.当前,利用AM真菌开展重金属污染土壤的生物修复已经引起环境学家和生态学家的广泛关注.基于此,围绕AM真菌在重金属污染土壤生物修复作用中的最新研究进展,从物理性防御体系的形成、对植物生理代谢的调控、生化拮抗物质的产生、基因表达的调控等角度探究AM真菌在重金属污染土壤生物修复中的作用机理,以期为利用AM真菌开展重金属污染的生物修复提供理论依据,并对本领域未来的发展和应用前景进行了展望.     

Understanding molecular mechanisms for improving phytoremediation of heavy metal-contaminated soils

Heavy metal pollution of soil is a significant environmental problem with a negative potential impact on human health and agriculture. Rhizosphere, as an important interface of soil and plants, plays a significant role in phytoremediation of contaminated soil by heavy metals, in which, microbial populations are known to affect heavy metal mobility and availability to the plant through release of chelating agents, acidification, phosphate solubilization and redox changes, and therefore, have potential to enhance phytoremediation processes. Phytoremediation strategies with appropriate heavy metal-adapted rhizobacteria or mycorrhizas have received more and more attention. In addition, some plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals, and they manage to survive under metal stresses. High tolerance to heavy metal toxicity could rely either on reduced uptake or increased plant internal sequestration, which is manifested by an interaction between a genotype and its environment.A coordinated network of molecular processes provides plants with multiple metal-detoxifying mechanisms and repair capabilities. The growing application of molecular genetic technologies has led to an increased understanding of mechanisms of heavy metal tolerance/accumulation in plants and, subsequently, many transgenic plants with increased heavy metal resistance, as well as increased uptake of heavy metals, have been developed for the purpose of phytoremediation. This article reviews advantages, possible mechanisms, current status and future direction of phytoremediation for heavy-metal–contaminated soils.     

Screening of plant growth promoting traits in heavy metals resistant bacteria: Prospects in phytoremediation

Phytoremediation is considered as a novel environmental friendly technology, which uses plants to remove or immobilize heavy metals. The use of metal-resistant plant growth-promoting bacteria (PGPB) constitutes an important technology for enhancing biomass production as well as tolerance of the plants to heavy metals. In this study, we isolated twenty seven (NF1-NF27) chromium resistant bacteria. The bacteria were tested for heavy metals (Cr, Zn, Cu, Ni, Pb and Co) resistance, Cr(VI) reduction and PGPB characters (phosphate solubilization, production of IAA and siderophores). The results showed that the bacterial isolates resist to heavy metals and reduce Cr(VI), with varying capabilities. 37.14% of the isolates have the capacity of solubilizing phosphate, 28.57% are able to produce siderophores and all isolates have the ability to produce IAA. Isolate NF2 that showed high heavy metal resistance and plant growth promotion characteristics was identified by 16S rDNA sequence analysis as a strain of Cellulosimicrobium sp.. Pot culture experiments conducted under greenhouse conditions showed that this strain was able to promote plant growth of alfalfa in control and in heavy metals (Cr, Zn and Cu) spiked soils and increased metal uptake by the plants. Thus, the potential of Cellulosimicrobium sp. for both bioremediation and plant growth promotion has significance in the management of environmental pollution.     

Application of polyphosphate metabolism to environmental and biotechnological problems

The synthesis and degradation of polyphosphate (polyP) are influenced by the energy state of the cell and extracellular phosphate levels. The import of excess phosphate and its incorporation into polyP under phosphate- and energy-rich growth conditions allows organisms to survive when phosphate or energy are depleted. Under phosphate-starvation conditions, phosphate can be recovered from polyP by hydrolysis. When the organism is energy starved, energy can be recovered either by regenerating the high-energy phosphoanhydride bond donor (ATP in most cases) or by hydrolysis of polyP and subsequent secretion of orthophosphate to recharge the transmembrane proton gradient. Understanding how the energy state of the cell and environmental phosphate levels affect polyP metabolism is essential to improving such environmental processes as enhanced biological phosphorus removal, a treatment process that is widely used to remove excess phosphate from wastewater. Manipulation of the genes responsible for polyP metabolism can also be used to improve gene expression from phosphate-starvation promoters and to remove heavy metals from contaminated environments.