title: Metabolite profiling to characterize disease-related bacteria: gluconate excretion by Pseudomonas aeruginosa mutants and clinical isolates from cystic fibrosis patients.
abstract: Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative σ factor RpoN (σ(54)), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.
authors.affiliation: Department of Surgery and Cancer, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Volker
authors.initials: V
authors.lastname: Behrends
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Bell
authors.forename: Manuel
authors.initials: M
authors.lastname: Liebeke
authors.forename: Anne
authors.initials: A
authors.lastname: Cordes-Blauert
authors.forename: Syedah N
authors.initials: SN
authors.lastname: Ashraf
authors.forename: Chandrika
authors.initials: C
authors.lastname: Nair
authors.forename: James E A
authors.initials: JE
authors.lastname: Zlosnik
authors.forename: Huw D
authors.initials: HD
authors.lastname: Williams
authors.forename: Jacob G
authors.initials: JG
authors.lastname: Bundy
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000382:microbiology* / Q000473:pathology; D024881:Drug Resistance, Bacterial / Q000502:physiology; D005260:Female; D005942:Gluconates / Q000378:metabolism*; D006801:Humans; D008297:Male; D055442:Metabolome; D011552:Pseudomonas Infections / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019105:Random Amplified Polymorphic DNA Technique
chemical_list: D001426:Bacterial Proteins; D005942:Gluconates; C030691:gluconic acid
keywords: Functional Genomics; Gene Regulation; Metabolic Footprinting; Metabolism; Metabolomics; Pseudomonas aeruginosa