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Metabolite Profiling to Characterize Disease-related Bacteria

V. Behrends,T. Bell,M. Liebeke,Anne Cordes-Blauert,S. N. Ashraf,C. Nair,J. Zlosnik,H. Williams,J. Bundy

Published 2013 in Journal of Biological Chemistry

ABSTRACT

Background: Phenotypic profiling of knockout libraries is a possible functional genomics strategy. Results: Gluconate excretion is a novel phenotype of the Pseudomonas aeruginosa rpoN mutant, which is also weakly associated with antibiotic susceptibility in a clinical strain panel. Conclusion: The rpoN phenotype results from catabolite repression deregulation of 6-phosphogluconate dehydratase. Significance: NMR profiling can uncover novel gene functions with potential clinical relevance. 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 (σ54), 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.

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