Intracellular redox status and antibiotic resistance in enterogastric micro-aerophilic bacteria: evidence for the 'scavenging of oxygen' hypothesis.
(2001)
Journal - Redox report : communications in free radical research (England )
Abstract :
Metronidazole and glutathione reduction activities were measured in situ in the micro-aerophilic bacteria Campylobacter coli and Helicobacter pylori employing 14N- and 1H-nuclear magnetic resonance spectroscopy. The properties of these enzyme activities were investigated in matched pairs of strains with sensitive and resistant phenotypes to the antimicrobial metronidazole. The results indicated that the ability of each type of strain to reduce metronidazole corresponded to its sensitive or resistance phenotype. Higher levels of glutathione reduction and a significantly lower Ki for metronidazole were observed in sensitive strains compared to resistant strains. These findings suggested a relationship between the cellular machinery regulating intracellular redox status in C. coli and H. pylori, and the effects of metronidazole on these bacteria, which supported the 'scavenging of oxygen' hypothesis.
| ISSN : | 1351-0002 |
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| Mesh Heading : | Campylobacter coli Drug Resistance Glutathione Helicobacter pylori Metronidazole Models, Biological Nuclear Magnetic Resonance, Biomolecular Oxidation-Reduction Oxygen metabolism metabolism metabolism |
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| Mesh Heading Relevant : | drug effects physiology metabolism drug effects pharmacology metabolism |
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In situ characterization of Helicobacter pylori arginase.
(1999)
Journal - Biochimica et biophysica acta (NETHERLANDS )
Abstract :
The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. One-dimensional 1H- and 13C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of l-arginine by the bacterium. Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates. A Km of 22+/-3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains. Divalent cations stimulated arginase activity, and the most potent activators were Co2+>Ni2+>Mn2+. The activity was highly specific for l-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin. The Ki of several inhibitors was measured and served also to characterize the enzyme activity. The presence of bicarbonate enhanced the hydrolysis of l-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties.
| ISSN : | 0006-3002 |
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| Mesh Heading : | Arginase Arginine Bacterial Proteins Biological Transport Cations, Divalent Enzyme Inhibitors Helicobacter pylori Isoelectric Point Kinetics Magnetic Resonance Spectroscopy Molecular Weight Ornithine Sequence Homology, Amino Acid Substrate Specificity metabolism chemistry pharmacology pharmacology pharmacology |
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| Mesh Heading Relevant : | chemistry enzymology |
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Purine metabolism and the microaerophily of Helicobacter pylori.
(1998)
Journal - Archives of microbiology (GERMANY )
Abstract :
The requirements for purine nucleotide synthesis, the effects of purine analogues, and the metabolism of adenine in the bacterium Helicobacter pylori were investigated employing cell culture techniques and one-dimensional NMR spectroscopy. Bacterial cells grew and proliferated in media lacking preformed purines, indicating that H. pylori can synthesize purine nucleotides de novo to meet its requirements. Blocking of this pathway in the absence of sufficient preformed purines for salvage nucleotide synthesis led to cell death. Analogues of purine nucleobases and nucleosides taken up by the cells were cytotoxic, suggesting that salvage routes could be exploited for therapy. Adenine or hypoxanthine were able to substitute for catalase in supporting cell growth and proliferation, suggesting a role for these bases in maintaining the microaerophilic conditions essentially required by the bacterium.
| ISSN : | 0302-8933 |
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| Mesh Heading : | Adenine Aminohydrolases Aminoimidazole Carboxamide Catalase Culture Media Drug Synergism Free Radical Scavengers Growth Inhibitors Helicobacter pylori Hydrogen Peroxide Hypoxanthine Oxygen Purine Nucleotides Purines pharmacology antagonists & inhibitors metabolism pharmacology pharmacology metabolism pharmacology pharmacology pharmacology drug effects enzymology pharmacology pharmacology metabolism antagonists & inhibitors antagonists & inhibitors metabolism |
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| Mesh Heading Relevant : | growth & development metabolism metabolism |
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Pyruvate metabolism in Campylobacter spp.
(1997)
Journal - Biochimica et biophysica acta (NETHERLANDS )
Abstract :
The metabolism of pyruvate by Campylobacter spp. was investigated employing one- and two-dimensional 1H, 13C and 31P nuclear magnetic resonance spectroscopy. Metabolically competent cells incubated aerobically with pyruvate yielded acetate, acetolactate, alanine, formate, lactate, and succinate. The production of acetolactate, alanine and lactate indicated the presence of acetohydroxy acid synthase, alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton to the Kreb's cycle, and the observation of pyruvate dehydrogenase activities in bacterial lysates supported this interpretation. Generation of pyruvate from L-serine in incubations with intact cells and lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was also formed in cell suspensions and lysates from phosphoenol pyruvate. The existence of anaplerotic sequences involving phosphoenol pyruvate carboxykinase and a malic enzyme were established in bacterial lysates. The activities of enzymes involved in the biosynthesis of isoleucine and valine were measured. Addition of pyruvate to different solid culture media inhibited bacterial growth, and the inhibition was attributed to the accumulation of acetate and formate. The variety of products formed using pyruvate as the sole substrate and the existence of anaplerotic sequences and anabolic pathways which employ pyruvate, showed the important role of this metabolite in the energy and biosynthesis metabolism of Campylobacter spp.
| ISSN : | 0006-3002 |
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| Mesh Heading : | Campylobacter Magnetic Resonance Spectroscopy Pyruvates growth & development methods |
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| Mesh Heading Relevant : | enzymology metabolism |
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Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein.
(1995)
Journal - European biophysics journal : EBJ (GERMANY )
Abstract :
The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and 31P, 13C, and 1H nuclear magnetic resonance spectroscopy (NMR), and electron magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5-, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and 13C- and 1H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% alpha-helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of alpha-helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles.
| ISSN : | 0175-7571 |
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| Mesh Heading : | Animals Cattle Chemistry, Physical Circular Dichroism Electron Spin Resonance Spectroscopy Lysophosphatidylcholines Magnetic Resonance Spectroscopy Myelin Basic Proteins Physicochemical Phenomena Protein Conformation Rabbits Scattering, Radiation Ultracentrifugation X-Rays |
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| Mesh Heading Relevant : | chemistry chemistry |
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Conformation of a tetradecapeptide epitope of myelin basic protein.
(1995)
Journal - European journal of biochemistry / FEBS (GERMANY )
Abstract :
The peptide AcAla-Ser-Gln-Lys-Arg-Pro-Ser-Gln-Arg-His-Gly-Ser-Lys-Tyr, which comprises the first 14 residues of the acetylated N-terminus of myelin basic protein, is an epitopic site for two monoclonal antibodies to the human protein. The conformations of the tetradecapeptide in aqueous solutions were investigated employing high-resolution 1H- and 13C-NMR spectroscopy. Two-dimensional techniques were used to assign the spectra observed from both nuclei. Nuclear-Overhauser-effect data, amide proton temperature coefficients, 13C spin-lattice relaxation times, distance geometry calculations and dynamic simulated annealing provided evidence that the solution conformations of the tetradecapeptide included a nascent alpha-helix in the N-terminal segment, and a loop extending from Ser7 to Ser12 that bring His10 and Tyr14 into close proximity.
| ISSN : | 0014-2956 |
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| Mesh Heading : | Amino Acid Sequence Animals Carbon Isotopes Epitopes Magnetic Resonance Spectroscopy Molecular Sequence Data Myelin Basic Proteins Protein Conformation Rabbits immunology |
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| Mesh Heading Relevant : | chemistry chemistry |
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Characterisation of glucose transport in Helicobacter pylori.
(1995)
Journal - Biochimica et biophysica acta (NETHERLANDS )
Abstract :
The nature of the glucose transport system in the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. Fast D-glucose uptake was demonstrated by using two methods of measuring glucose transport. The transport of 2-deoxy-D-glucose was inhibited competitively by D-glucose; and the efflux of 2-deoxy-D-glucose from cells also was affected by the presence of D-glucose. The transport of 2-deoxy-D-glucose was saturable with a Km of 4.8 mM and Vmax of 146.6 pmol (microliter cell water)-1 at 20 degrees C. The transport was temperature-dependent with energies of activation of 6.8 and 51.0 kJ mol-1 for 0.2 and 20 mM 2-deoxy-D-glucose, respectively. The temperature dependence and saturable nature of transport suggested the presence of one or more glucose carriers. The substrate specificity of the transport system was studied by measuring the effects of mono- and disaccharides on the rates of transport of the glucose analogue. The most significant inhibitory effects were obtained with D-galactose and L-arabinose. Lack of transport inhibition by L-glucose established the stereospecificity of the transporters for the D-isomer of glucose. Higher rates of 2-deoxy-D-glucose transport were measured in the presence of sodium ions than for other monovalent cations, and the presence of amiloride inhibited transport of the monosaccharide. No inhibition was observed with cytochalasin B, phloretin or phloridzin. The results suggested the existence of specific D-glucose transporters and that the glucose transport system of H. pylori is significantly different from other known bacterial transporters.
| ISSN : | 0006-3002 |
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| Mesh Heading : | Amiloride Arabinose Biological Transport Cations, Monovalent Cytochalasin B Deoxyglucose Galactose Glucose Helicobacter pylori Kinetics Phloretin Phlorhizin Sodium Temperature Thermodynamics Valinomycin pharmacology pharmacology pharmacology pharmacology pharmacology drug effects pharmacology pharmacology pharmacology pharmacology |
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| Mesh Heading Relevant : | metabolism metabolism |
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Fumarate reductase: a target for therapeutic intervention against Helicobacter pylori.
(1995)
Journal - Archives of biochemistry and biophysics (UNITED STATES )
Abstract :
The potential of fumarate reductase as a therapeutic target against the human pathogen Helicobacter pylori was investigated by studying the cytotoxicity of morantel, oxantel, and thiabendazole, known to inhibit the enzyme in parasitic worms. Nuclear magnetic resonance spectroscopy was employed to investigate the effects of the inhibitors on the fumarate reductase activity of laboratory-adapted and wild-type bacterial strains. Production of succinate from fumarate in H. pylori cells was inhibited by morantel, oxantel, and thiabendazole. Cell growth and viability techniques were used to examine the bacteriostatic and bactericidal effects of the three anthelmintics. Each of the antiparasites arrested growth and produced cell death in liquid cultures, although the minimal inhibitory and bactericidal concentrations of these compounds are such that they would not be of therapeutic use. The strength of the effects as measured by minimal inhibitory and bactericidal concentrations was oxantel > thiabendazole > morantel. The findings suggested that fumarate reductase is an essential component of the metabolism of H. pylori and as such constitutes a possible target for therapeutic intervention in the treatment of the bacterium.
| ISSN : | 0003-9861 |
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| Mesh Heading : | Aconitate Hydratase Anti-Bacterial Agents Fumarate Hydratase Helicobacter Infections Helicobacter pylori Humans Kinetics Malate Dehydrogenase Microbial Sensitivity Tests Morantel Pyrantel Succinate Dehydrogenase Thiabendazole metabolism toxicity metabolism enzymology growth & development metabolism therapeutic use therapeutic use toxicity therapeutic use |
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| Mesh Heading Relevant : | drug therapy drug effects toxicity analogs & derivatives antagonists & inhibitors toxicity |
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De novo synthesis of pyrimidine nucleotides by Helicobacter pylori.
(1994)
Journal - The Journal of applied bacteriology (ENGLAND )
Abstract :
The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.
| ISSN : | 0021-8847 |
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| Mesh Heading : | Aspartate Carbamoyltransferase Aspartic Acid Bicarbonates Deoxycytidine Kinase Helicobacter pylori Isotope Labeling Ligases Magnetic Resonance Spectroscopy Orotate Phosphoribosyltransferase Pentosyltransferases Phosphorus Isotopes Pyrimidine Nucleotides analysis metabolism metabolism analysis enzymology analysis analysis analysis |
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| Mesh Heading Relevant : | Carbon-Nitrogen Ligases metabolism biosynthesis |
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Pyruvate metabolism in Helicobacter pylori.
(1994)
Journal - Archives of microbiology (GERMANY )
Abstract :
The metabolism of pyruvate by Helicobacter pylori was investigated employing one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. Generation of pyruvate from L-serine in incubations with whole cell lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was formed also in cell suspensions and lysates from phosphoenol pyruvate. Metabolically competent cells incubated aerobically with pyruvate yielded alanine, lactate, acetate, formate, and succinate. The production of alanine and lactate indicated the presence of alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed-acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton into the Kreb's cycle. Addition of pyruvate to various liquid culture media did not affect bacterial growth or loss of viability. The variety of products formed using pyruvate as the sole substrate showed the important role of this metabolite in the energy metabolism of H. pylori.
| ISSN : | 0302-8933 |
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| Mesh Heading : | Acetic Acid Acetic Acids Anaerobiosis Animals Culture Media Energy Metabolism Fermentation Formic Acids Helicobacter pylori Humans Magnetic Resonance Spectroscopy Oxidation-Reduction Pyruvates Pyruvic Acid Succinates Succinic Acid metabolism metabolism growth & development metabolism |
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| Mesh Heading Relevant : | metabolism metabolism |
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Fluorinated probes to measure carboxylesterase activities using 19F NMR spectroscopy: application to erythrocytes and Helicobacter pylori.
(1993)
Journal - Archives of biochemistry and biophysics (UNITED STATES )
Abstract :
Eight fluorinated compounds were tested as putative probes to measure carboxylesterase activity employing 19F nuclear magnetic resonance spectroscopy. The method takes advantage of the sensitivity of fluorine resonances to the changes in the chemical bonding in the covalent structures where they are located. Determination of the kinetic parameters of ethyl fluoroacetate and diethyl fluoromalonate in hemolysates showed that these probes were well suited to study carboxylesterase activities in complex systems the potential of these probes for noninvasive applications was demonstrated in measurements of carboxylesterase kinetic parameters in intact erythrocytes. The presence of carboxylesterases was established in several strains of the bacterium Helicobacter pylori employing 19F nuclear magnetic resonance spectroscopy, and the kinetic parameters of these enzymes for ethyl fluoroacetate and diethyl fluoromalonate were measured.
| ISSN : | 0003-9861 |
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| Mesh Heading : | Animals Carboxylic Ester Hydrolases Erythrocytes Fluorine Helicobacter pylori Humans Kinetics Liver Rabbits enzymology |
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| Mesh Heading Relevant : | analysis enzymology enzymology |
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Glucose phosphorylation in Helicobacter pylori.
(1993)
Journal - Archives of biochemistry and biophysics (UNITED STATES )
Abstract :
Saccharide kinase activities in Helicobacter pylori were investigated by incubating bacterial lysates with ATP and mono- or disaccharides and monitoring directly the appearance of phosphorylated products using 13C or 31P nuclear magnetic resonance spectroscopy. The monosaccharides employed included two trioses, two tetroaldoses, one tetroketose, five aldopentoses, two ketopentoses, five aldohexoses, three ketohexoses, and gluconic and glucuronic acids; the disaccharides studied were maltose, trehalose, cellobiose, sucrose, lactose, gentobiose, and melibiose. D-Glucose was the only sugar phosphorylated among all the carbohydrates examined. The kinase activity of lysates was studied by measuring the rates of formation of glucose 6-phosphate. The substrate specificity, the relatively high KM, and the absence of substrate inhibition suggested that the enzyme is a glucokinase rather than a hexokinase. Most of the glucose kinase activity was observed with the pellet fraction obtained by centrifugation, suggesting an association of the enzyme with the bacterial cell envelope.
| ISSN : | 0003-9861 |
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| Mesh Heading : | Adenosine Triphosphate Carbon Isotopes Disaccharides Glucokinase Glucose Glucose-6-Phosphate Glucosephosphates Helicobacter pylori Kinetics Magnetic Resonance Spectroscopy Monosaccharides Phosphorus Phosphorylation metabolism metabolism metabolism metabolism enzymology methods metabolism |
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| Mesh Heading Relevant : | metabolism metabolism |
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Metabolism and Genetics of Helicobacter pylori: the Genome Era
(1999)
Journal - Microbiology and Molecular Biology Reviews
Abstract :
The publication of the complete sequence of Helicobacter pylori 26695 in 1997 and more recently that of strain J99 has provided new insight into the biology of this organism. In this review, we attempt to analyze and interpret the information provided by sequence annotations and to compare these data with those provided by experimental analyses. After a brief description of the general features of the genomes of the two sequenced strains, the principal metabolic pathways are analyzed. In particular, the enzymes encoded by H. pylori involved in fermentative and oxidative metabolism, lipopolysaccharide biosynthesis, nucleotide biosynthesis, aerobic and anaerobic respiration, and iron and nitrogen assimilation are described, and the areas of controversy between the experimental data and those provided by the sequence annotation are discussed. The role of urease, particularly in pH homeostasis, and other specialized mechanisms developed by the bacterium to maintain its internal pH are also considered. The replicational, transcriptional, and translational apparatuses are reviewed, as is the regulatory network. The numerous findings on the metabolism of the bacteria and the paucity of gene expression regulation systems are indicative of the high level of adaptation to the human gastric environment. Arguments in favor of the diversity of H. pylori and molecular data reflecting possible mechanisms involved in this diversity are presented. Finally, we compare the numerous experimental data on the colonization factors and those provided from the genome sequence annotation, in particular for genes involved in motility and adherence of the bacterium to the gastric tissue.
A Novel Mechanism for Resistance to the Antimetabolite N-Phosphonoacetyl-l-Aspartate by Helicobacter pylori
(1998)
Journal - Journal of Bacteriology
Abstract :
The mechanism of resistance to N-phosphonoacetyl-l-aspartate (PALA), a potent inhibitor of aspartate carbamoyltransferase (which catalyzes the first committed step of de novo pyrimidine biosynthesis), in Helicobacter pylori was investigated. At a 1 mM concentration, PALA had no effects on the growth and viability of H. pylori. The inhibitor was taken up by H. pylori cells and the transport was saturable, with a Km of 14.8 mM and a Vmax of 19.1 nmol min-1 µl of cell water-1. By 31P nuclear magnetic resonance (NMR) spectroscopy, both PALA and phosphonoacetate were shown to have been metabolized in all isolates of H. pylori studied. A main metabolic end product was identified as inorganic phosphate, suggesting the presence of an enzyme activity which cleaved the carbon-phosphorus (C-P) bonds. The kinetics of phosphonate group cleavage was saturable, and there was no evidence for substrate inhibition at higher concentrations of either compound. C-P bond cleavage activity was temperature dependent, and the activity was lost in the presence of the metal chelator EDTA. Other cleavages of PALA were observed by 1H NMR spectroscopy, with succinate and malate released as main products. These metabolic products were also formed when N-acetyl-l-aspartate was incubated with H. pylori lysates, suggesting the action of an aspartase. Studies of the cellular location of these enzymes revealed that the C-P bond cleavage activity was localized in the soluble fraction and that the aspartase activity appeared in the membrane-associated fraction. The results suggested that the two H. pylori enzymes transformed the inhibitor into noncytotoxic products, thus providing the bacterium with a mechanism of resistance to PALA toxicity which appears to be unique.