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.     

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.     

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.     

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.     

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.     

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.     

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.     

Inorganic polyphosphate is required for motility of bacterial pathogens

The ppk gene encodes polyphosphate kinase (PPK), the principal enzyme in many bacteria responsible for the synthesis of inorganic polyphosphate (polyP) from ATP. A null mutation in the ppk gene of six bacterial pathogens renders them greatly impaired in motility on semisolid agar plates; this defect can be corrected by the introduction of ppk gene in trans. In view of the fact that the motility of pathogens is essential to invade and establish systemic infections in host cells, this impairment in motility suggests a crucial and essential role of PPK or polyP in bacterial pathogenesis.     

Polyphosphate kinase protects Salmonella enterica from weak organic acid stress

Mutants of Salmonella enterica lacking polyphosphate kinase (ppk) grow poorly in the presence of the weak organic acids acetate, propionate, and benzoate. This sensitivity is corrected by methionine and seems to result from destabilization of MetA (homoserine transsuccinylase), the first enzyme in methionine biosynthesis. The MetA protein is known to be sensitive to thermal inactivation, and ppk mutants are more sensitive to heat-induced methionine auxotrophy. Peroxide increases the sensitivity of ppk mutants to both heat and acid and may oxidatively damage (carbonylate) destabilized MetA. While acid appears to impair methionine biosynthesis, it leads to derepression of MetA and may inhibit growth by causing toxic accumulation of denatured protein. This is supported by the observation that the overexpression of MetA in ppk mutants causes acid sensitivity that is not corrected by methionine. We propose that polyphosphate acts as a chemical chaperone that helps refold MetA and/or may stimulate proteolysis of toxic denatured protein. The instability of MetA protein may provide a metabolic fuse that blocks growth under conditions that denature proteins; the sensitivity of this fuse is modulated by polyphosphate.     

NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum

Corynebacterium glutamicum is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes, ppk2A (corresponds to NCgl0880) and ppk2B (corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of ppk2B decreased PPK activity and cellular poly P content, while overexpression of ppk2B increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of ppk2A changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of C. glutamicum is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with (31)P nuclear magnetic resonance. The catalytic efficiency of C. glutamicum PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The ppk2B deletion mutant, which accumulated very little poly P and grew as C. glutamicum wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.     

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.     

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction

An Escherichia coli strain was generated by fusion of a merA-deleted broad-spectrum mer operon from Pseudomonas K-62 with a bacterial polyphosphate kinase gene (ppk) from Klebsiella aerogenes in vector pUC119. A large amount of the ppk-specified polyphosphate was identified in the mercury-induced bacterium with the fusion plasmid designated pMKB18 but not in the cells without mercury induction. These results suggest that the synthesis of polyphosphate as well as the expression of the mer genes is mercury-inducible and regulated by merR. The E. coli strain with pMKB18 was more resistant to both Hg2+ and C6H5Hg+ than its isogenic strain with cloning vector pUC119. The recombinant strain accumulated more mercury from Hg2+- and C6H5Hg+-contaminated medium. Hg2+ transported into the cytoplasm appeared to be bound by chelation with the polyphosphate produced by the recombinant cells. The transported phenylmercury was degraded to Hg2+ before the chelation since polyphosphate did not directly chelate with C6H5Hg+. These results indicate that polyphosphate is capable of reducing the cytotoxicity of the transported Hg2+ probably via chelation between polyphosphate and Hg2+.     

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

Background

Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence.

Findings

By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin.

Conclusions

Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0012-0) contains supplementary material, which is available to authorized users.     

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。     

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).     

Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli.

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase.