Alginate, inorganic polyphosphate, GTP and ppGpp synthesis co-regulated in Pseudomonas aeruginosa: implications for stationary phase survival and synthesis of RNA/DNA precursors

The regulatory protein AlgR2 in Pseudomonas aeruginosa positively regulates nucleoside diphosphate kinase (Ndk) and succinyl-CoA synthetase, enzymes critical in nucleoside triphosphate (NTP) formation. AlgR2 positively regulates the production of alginate, GTP, ppGpp and inorganic polyphosphate (poly P). An algR2 mutant with low levels of these metabolites has them restored by introducing and overexpressing either the algR2 or the ndk gene into the algR2 mutant. Thus, Ndk is involved in the formation of these compounds and largely prevents the death of the algR2 mutant, which occurs early in the stationary phase. We demonstrate that the 12 kDa Ndk–pyruvate kinase (Pk) complex, previously shown to generate predominantly GTP instead of all the NTPs, has a low affinity for the deoxynucleoside diphosphates and cannot generate the dNTPs needed for DNA replication and cell division; this complex may thus be involved in regulating the levels of both NTPs and dNTPs that modulate cell division and survival in the stationary phase.     

The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

We report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis . The N-terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli . Immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4B matrix and passing the total cell extract through it revealed four proteins (P₇₀, P₆₅, P₆₀, and P₅₀, respectively) of M _r 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M _r 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P₇₀ protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.     

Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis.

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.     

Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDP<-->ADP+(d)NTP. This reaction, suggested to occur by the transfer of the gamma-phosphoryl from ATP to the nucleoside diphosphate, is overall similar to that normally carried out by nucleoside diphosphate kinase (Ndk). Accordingly, Adk was proposed to be responsible for residual Ndk-like activity measured in a mutant strain of Escherichia coli, where the ndk gene was disrupted. We present data supporting a mechanism for the synthesis of nucleoside triphosphates by Adk that unlike the previously suggested mechanism mentioned above are in complete agreement with the current knowledge about the Adk enzyme and its various catalytic properties. We propose that nucleoside triphosphate synthesis occurs by beta-phosphoryl transfer from ADP to any bound nucleoside diphosphate. Our results point to the fact that the proposed Ndk-like mechanism of Adk originated from an erroneous interpretation of data, in that contamination of ATP preparations with AMP and ADP was not taken into account. Our results also address the proposed role of Adk in restoring a normal growth rate of mutant strains of E. coli lacking Ndk. These mutant strains apparently, in spite of a mutator phenotype, are able to synthesise nucleoside triphosphates by alternative pathways to maintain the same growth rate as the wildtype.     

Succinyl coenzyme A synthetase of Pseudomonas aeruginosa with a broad specificity for nucleoside triphosphate (NTP) synthesis modulates specificity for NTP synthesis by the 12-kilodalton form of nucleoside diphosphate kinase

Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the sucCD operon encoding the alpha and beta subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P(i)), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid P. aeruginosa.     

Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.     

Double-stranded RNA bacteriophage phi 6 protein P4 is an unspecific nucleoside triphosphatase activated by calcium ions.

Double-stranded RNA bacteriophage phi 6 has an envelope surrounding the nucleocapsid (NC). The NC is composed of a surface protein, P8, and proteins P1, P2, P4, and P7, which form a dodecahedral polymerase complex enclosing the segmented viral genome. Empty polymerase complex particles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphosphates (NTPs). Photoaffinity labelling of both the NC and the procapsid has earlier been used to show that ATP binds to protein P4 and that the NC hydrolyzes NTPs. Using the NC and the NC core particles (NCs lacking surface protein P8) and purified protein P4, we demonstrate here that multimeric P4 is the active NTPase. Isolation of multimeric P4 is successful only in the presence of NTPs. The activity of P4 is the same in association with the viral particles as it is in pure form. P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NTPs to the corresponding nucleoside diphosphates. The Km of the reaction for ATP, GTP, and UTP is around 0.2 to 0.3 mM. The NTP hydrolysis by P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM. Competition experiments indicate that Mg2+ and Ca2+ ions have approximately equal binding affinities for P4. They might compete for a common binding site. The nucleotide specificity and enzymatic properties of the P4 NTPase are similar to the NTP hydrolysis reaction conditions needed to translocate and condense the viral positive-sense RNAs to the procapsid particle.     

Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2an6 algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction     

Regulation of nucleoside diphosphate kinase and an alternative kinase in Escherichia coli: role of the sspA and rnk genes in nucleoside triphosphate formation

We have previously reported that two genes cloned from a cosmid library of Escherichia coli can restore mucoidy to an algR2 mutant of Pseudomonas aeruginosa . AlgR2 is a protein involved in the regulation of nucleoside diphosphate kinase (Ndk) as well as alginate synthesis in P. aeruginosa . One of the E. coli genes, rnk , encodes a 14.9 kDa protein with no homology to any other proteins. The other gene, sspA , encodes the stringent starvation protein, a regulatory protein involved in stationary-phase regulation and the stringent response of E. coli . While both rnk and sspA restored alginate production to the P. aeruginosa algR2 mutant, only rnk restored Ndk activity to the mutant. In this report, we have examined the effect of mutations in rnk and sspA on the levels of Ndk in E. coli . We find that a mutation in rnk drastically reduces the level of Ndk in E. coli . A mutation in sspA , however, affects the level of another nucleoside diphosphate kinase distinct from Ndk. The proteins can be easily distinguished from each other by their different affinities for nucleoside diphosphates (NDPs) and also by the differential effect of anti-Ndk antibodies on the reactions they catalyse. The ability of either of these two proteins to restore alginate synthesis in the algR2 mutant of P. aeruginosa demonstrates the importance of nucleoside triphosphate synthesis and energy metabolism for alginate synthesis. Additionally, a role for the stringent starvation protein (SspA) in the modulation of nucleoside triphosphate (NTP) levels in E. coli is also suggested from these experiments.     

The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates

Mollicutes are wall-less bacteria and cause various diseases in humans, animals and plants. They have the smallest genomes with low G + C content and lack many genes of DNA, RNA and protein precursor biosynthesis. Nucleoside diphosphate kinase (NDK), a house-keeping enzyme that plays a critical role in the synthesis of nucleic acids precursors, i.e. NTPs and dNTPs, is absent in all the Mollicutes genomes sequenced to date. Therefore, it would be of interest to know how Mollicutes synthesize dNTPs/NTPs without NDK. To answer this question, nucleoside monophosphate kinases (NMPKs) from Ureaplasma were studied regarding their role in the synthesis of NTPs/dNTPs. In this work, Ureaplasma adenylate kinase, cytidylate kinase, uridylate kinase and thymidylate kinase were cloned and expressed in Escherichia coli. The recombinant enzymes were purified and characterized. These NMPKs are base specific, as indicated by their names, and capable of converting (d)NMPs directly to (d)NTPs. The catalytic rates of (d)NTPs and (d)NDP synthesis by these NMPKs were determined using tritium-labelled (d)NMPs, and the rates for (d)NDP synthesis, in general, were much higher (up to 100-fold) than that of (d)NTP. Equilibrium studies with adenylate kinase suggested that the rates of NTPs/dNTPs synthesis by NMPKs in vivo are probably regulated by the levels of (d)NMPs. These results strongly indicate that NMPKs could substitute the NDK function in vivo.     

Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa: roles of algR2 and algH.

Alginate is an important virulence factor for Pseudomonas aeruginosa during infection of the lungs of cystic fibrosis patients. The genes encoding enzymes for alginate production by P. aeruginosa are normally silent. They are activated in response to several environmental conditions, including high osmolarity, exposure to ethanol, or long-term growth under conditions of nutrient deprivation. Several genes which participate in the activation of alginate gene promoters have been identified; among these is the algR2 (algQ) gene. AlgR2 is an 18-kDa protein which has been shown to regulate the critical algD gene encoding GDP-mannose dehydrogenase as well as to regulate the levels of a tricarboxylic acid cycle enzyme, i.e., succinyl coenzyme A synthetase, and nucleoside diphosphate kinase (Ndk), an enzyme involved in nucleoside triphosphate synthesis. Succinyl coenzyme A synthetase and Ndk form a complex in P. aeruginosa. While algR2 is required for alginate synthesis at 37 degrees C, an algR2 insertion mutant was still able to make alginate slowly at 37 or at 30 degrees C. We used this observation to identify and clone a gene, termed algH. A strain with mutations in both algR2 and algH is unable to produce alginate at either 37 or 30 degrees C, and it is fully defective in Ndk production.     

Comparison between ATP-supported and GTP-supported phosphate turnover of the calcium-transporting sarcoplasmic reticulum membranes.

The study deals with the interrelationship of the phosphate-transferring activities of the calcium-transporting sarcoplasmic reticulum membrane vesicles: the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the calcium-dependent NTase, and the phosphorylation of NDP by inorganic phosphate in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. It is demonstrated for the phosphate transfer from ITP to GDP that the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the nucleoside diphosphate and triphosphate species are deduced according to this mechanism. The enzyme's affinity for the nucleoside triphosphates and diphosphates depends on its functional state being considerably lower under conditions of NTP-NDP exchange than during NTP splitting or NTP synthesis. ATP and GTP are split with the same low rates when calcium-activated NTPase is inhibited by high internal calcium concentrations after calcium transport has reached steady state. The rates of the NTP-NDP exchange reactions, however, differ by a factor of about 10 being approximately equal to 3 mumol . mg-1 . min-1 for ATP-ADP and only approximately equal to 0.3 mumol . mg-1 . min-1 (22 degrees C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made calcium-permeable, the calcium transport ATPase is turned on and the rates of GTP and ATP splitting increase about tenfold. Yet, while the rate of ATP-ADP exchange is little reduced, the rate of GTP-GDP exchange drops by approximately 50%. The persisting exchange activity of calcium-permeable vesicles demonstrates that high internal calcium concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.     

Distinct interactions of GTP,UTP, and CTP with G(s) proteins

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Mammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α-, β- or the γ-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk–G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α-, β- and γ-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α- and γ-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.     

Fuel Specificity of the Hepatitis C Virus NS3 Helicase

The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3′-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson-Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2′ and 3′ carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn²⁺ supporting about 10 times faster unwinding than Mg²⁺. Unlike Mg²⁺, Mn²⁺ does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.     

A method for concentration of nucleoside triphosphates by coprecipitation with calcium fluoride

A method for concentration of nucleoside triphosphates (NTP) is described. NTPs are quantitatively coprecipitated from the solution with calcium fluoride. The precipitate is separated by filtration through a membrane filter and NTPs are dissolved from the filter by immersing it in 0.5 N H2SO4. With this method also nucleoside diphosphates can be efficiently concentrated, but the method does not work with nucleoside monophosphates or cyclic AMP.     

Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis

Elastase is a major virulence factor in Pseudomonas aeruginosa that is believed to cause extensive tissue damage during infection in the human host. Elastase is secreted in non-mucoid P. aeruginosa. It is known that secretion of most virulence factors such as elastase, lipase, exotoxin A, etc., in P. aeruginosa is greatly reduced in alginate-secreting mucoid cells isolated from the lungs of cystic fibrosis (CF) patients. We have previously reported that in mucoid P. aeruginosaan intracellular protease cleaves the 16 kDa form of nucleoside diphosphate kinase (Ndk) to a truncated 12 kDa form. This smaller form is membrane associated and has been observed to form complexes with specific proteins to predominantly generate GTP, an important molecule in alginate synthesis. The main aim of this study was to purify and characterize this protease. The protease was purified by hydrophobic interaction chromatography of the crude extract of mucoid P. aeruginosa 8821, a CF isolate. Further analysis using a gelatin containing SDS–polyacrylamide gel detected the presence of a 103 kDa protease, which when boiled, migrated as a 33 kDa protein on a SDS–polyacrylamide gel. The first 10 amino acids from the N-terminus of the 33 kDa protease showed 100% identity to the mature form of elastase. An elastase-negative lasB ::Cm knock-out mutant in the mucoid 8821 background was constructed, and it showed a non-mucoid phenotype. This mutant showed the presence of only the 16 kDa form of Ndk both in the cytoplasm and membrane fractions. We present evidence for the retention of active elastase in the periplasm of mucoid P. aeruginosa and its role in the generation of the 12 kDa form of Ndk. Finally, we demonstrate that elastase, when overproduced in both mucoid and non-mucoid cells, stimulates alginate synthesis. This suggests that the genetic rearrangements that trigger mucoidy in P. aeruginosa also allow retention of elastase in the periplasm in an active oligomeric form that facilitates cleavage of 16 kDa Ndk to its 12 kDa form for the generation of GTP, required for alginate synthesis.     

Characterization of the interaction of wheat germ protein synthesis initiation factor eIF-3 with mRNA oligonucleotide and cap analogues.

Direct fluorescence titration experiments of wheat germ protein synthesis initiation factor eIF-3 with mRNA cap and oligoribonucleotide analogues were performed in order to determine the equilibrium association constants (Keq) for the eIF-3.mRNA interaction as a function of pH and temperature. These data suggest that (i) the eIF-3.mRNA interaction is not cap-specific (i.e., m7G-specific), (ii) ATP hydrolysis is not involved in the interaction, and (iii) the interaction is primarily ionic in nature. Competition experiments between a rabbit alpha-globin mRNA oligoribonucleotide analogue and either mRNA cap analogues or nucleoside triphosphates (NTPs) are also reported; these experiments indicate that NTPs act as both activators and competitive inhibitors of the mRNA.eIF-3 association. The results are consistent with a partially uncompetitive binding mechanism, whereby at low NTP concentrations (less than or equal to 10 microM) the bound NTP enhances subsequent mRNA binding to eIF-3, perhaps by inducing a conformational change, and at higher NTP concentrations, the NTP acts as a competitive inhibitor for the mRNA binding site on eIF-3.     

Nucleotide binding by the poliovirus RNA polymerase.

Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes. The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme. Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs. Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with [32P]GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP. Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography. In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column. Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP. Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss. These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.