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.     

Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.     

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.     

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.     

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+.     

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.     

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.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.     

SOS system induction in Escherichia coli cells with distinct levels of ribonucleotide reductase activity.

The UV-mediated induction of recA and sfiA genes in Escherichia coli cells with distinct levels of dATP has been studied. Low levels of dATP were obtained by using either a temperature-sensitive ribonucleotide (RDP) reductase-deficient (nrdA) mutant or a wild-type strain treated with hydroxyurea. High pools of dATP were achieved by using a plasmid overproducing RDP reductase. The results obtained show that expression of the recA and sfiA genes was inhibited neither in the UV-irradiated nrdA mutant at 42 degrees C nor in the wild-type strain in the presence of hydroxyurea. Likewise, the increase of the dATP pool did not enhance recA and sfiA gene expression after UV irradiation. All these data suggest that the basal level of dATP is not a limiting factor in the process of induction of the SOS system in Escherichia coli.     

Diverse phenotypes resulting from polyphosphate kinase gene (ppk1) inactivation in different strains of Helicobacter pylori

Connections among biochemical pathways should help buffer organisms against environmental stress and affect the pace and trajectory of genome evolution. To explore these ideas, we studied consequences of inactivating the gene for polyphosphate kinase 1 (ppk1) in strains of Helicobacter pylori, a genetically diverse gastric pathogen. The PPK1 enzyme catalyzes synthesis of inorganic polyphosphate (poly P), a reservoir of high-energy phosphate bonds with multiple roles. Prior analyses in less-fastidious microbes had implicated poly P in stress resistance, motility, and virulence. In our studies, ppk1 inactivation caused the expected near-complete absence of poly P (>250-fold decrease) but had phenotypic effects that differed markedly among unrelated strains: (i) poor initial growth on standard brain heart infusion agar (five of six strains tested); (ii) weakened colonization of mice (4 of 5 strains); (iii) reduced growth on Ham's F-12 agar, a nutritionally limiting medium (8 of 11 strains); (iv) heightened susceptibility to metronidazole (6 of 17 strains); and (v) decreased motility in soft agar (1 of 13 strains). Complementation tests confirmed that the lack of growth of one Deltappk1 strain on F-12 agar and the inability to colonize mice of another were each due to ppk1 inactivation. Thus, the importance of ppk1 to H. pylori differed among strains and the phenotypes monitored. We suggest that quantitative interactions, as seen here, are common among genes that affect metabolic pathways and that H. pylori's high genetic diversity makes it well suited for studies of such interactions, their underlying mechanisms, and their evolutionary consequences.     

Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-deficient strain remains viable

Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmdeltaRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmdeltaRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2-151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only first 151 amino acids of the RecA protein. This S. rimiosus rec A mutant can therefore be considered a completely RecA-deficient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way.     

Magnetic resonance-based visualization of gene expression in mammalian cells using a bacterial polyphosphate kinase reporter gene

Gene expression reporter systems, in which a promoter of interest is cloned upstream of a readily assayed reporter gene, have been developed and used extensively to study gene expression in prokaryotes and eukaryotes. Unfortunately, most of these systems cannot be used to assay gene expression in nonsuperficial tissues in living organisms. This study examines a novel reporter gene system based on the gene encoding Escherichia coli polyphosphate kinase (PPK), which can be used to monitor gene expression in mammalian cells. PPK catalyzes the synthesis of inorganic polyphosphate (polyP) from ATP, and because mammalian cells do not contain detectable levels of polyP, PPK activity can be measured in mammalian cells using 31P-magnetic resonance spectroscopy or 31P-magnetic resonance imaging. The ppk reporter gene system described here is noninvasive, does not require an exogenous substrate, and can potentially be used in internal tissues of living organisms.     

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.     

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.     

Protection of nonmodified phageλ againstEcoK restriction mediated byrecA protein

A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions. The host cells used were E. coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir). An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that the recA protein protects phage DNA during the process of restriction. A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth. By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.     

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.     

Inorganic polyphosphate and polyphosphate kinase: their novel biological functions and applications

In this review, we discuss the following two subjects: 1) the physiological function of polyphosphate (poly(P)) as a regulatory factor for gene expression in Escherichia coli, and 2) novel functions of E. coli polyphosphate kinase (PPK) and their applications. With regard to the first subject, it has been shown that E. coli cells in which yeast exopolyphosphatase (poly(P)ase), PPX1, was overproduced reduced resistance to H2O2 and heat shock as did a mutant whose polyphosphate kinase gene is disrupted. Sensitivity to H2O2 and heat shock evinced by cells that overproduce PPX1 is attributed to depressed levels of rpoS expression. Since rpoS is a central element in a regulatory network that governs the expression of stationary-phase-induced genes, poly(P) affects the expression of many genes through controlling rpoS expression. Furthermore, poly(P) is also involved in expression of other stress-inducible genes that are not directly regulated by rpoS. The second subject includes the application of novel functions of PPK for nucleoside triphosphate (NTP) regeneration. Recently E. coli PPK has been found to catalyze the kination of not only ADP but also other nucleoside diphosphates using poly(P) as a phospho-donor, yielding NTPs. This nucleoside diphosphate kinase-like activity of PPK was confirmed to be available for NTP regeneration essential for enzymatic oligosaccharide synthesis using the sugar nucleotide cycling method. PPK has also been found to express a poly(P):AMP phosphotransferase activity by coupling with adenylate kinase (ADK) in E. coli. The ATP-regeneration system consisting of ADK, PPK, and poly(P) was shown to be promising for practical utilization of poly(P) as ATP substitute.     

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.