title: Metabolomics Analysis Reveals the Participation of Efflux Pumps and Ornithine in the Response of Pseudomonas putida DOT-T1E Cells to Challenge with Propranolol.
abstract: Efflux pumps are critically important membrane components that play a crucial role in strain tolerance in Pseudomonas putida to antibiotics and aromatic hydrocarbons that result in these toxicants being expelled from the bacteria. Here, the effect of propranolol on P. putida was examined by sudden addition of 0.2, 0.4 and 0.6 mg mL-1 of this β-blocker to several strains of P. putida, including the wild type DOT-T1E and the efflux pump knockout mutants DOT-T1E-PS28 and DOT-T1E-18. Bacterial viability measurements reveal that the efflux pump TtgABC plays a more important role than the TtgGHI pump in strain tolerance to propranolol. Mid-infrared (MIR) spectroscopy was then used as a rapid, high-throughput screening tool to investigate any phenotypic changes resulting from exposure to varying levels of propranolol. Multivariate statistical analysis of these MIR data revealed gradient trends in resultant ordination scores plots, which were related to the concentration of propranolol. MIR illustrated phenotypic changes associated with the presence of this drug within the cell that could be assigned to significant changes that occurred within the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during the growth of bacteria following toxic perturbation with the same concentration levels of propranolol. Metabolic profiling revealed that ornithine, which was only produced by P. putida cells in the presence of propranolol, presents itself as a major metabolic feature that has important functions in propranolol stress tolerance mechanisms within this highly significant and environmentally relevant species of bacteria.
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: Ali
authors.initials: A
authors.lastname: Sayqal
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: Drupad K
authors.initials: DK
authors.lastname: Trivedi
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: Najla
authors.initials: N
authors.lastname: AlMasoud
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: David I
authors.initials: DI
authors.lastname: Ellis
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: Nicholas J W
authors.initials: NJ
authors.lastname: Rattray
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
mesh_terms: D002244:Carbon / Q000378:metabolism; D016002:Discriminant Analysis; D008401:Gas Chromatography-Mass Spectrometry; D019450:Genes, MDR; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D009952:Ornithine / Q000378:metabolism*; D025341:Principal Component Analysis; D011433:Propranolol / Q000494:pharmacology*; D016958:Pseudomonas putida / Q000166:cytology* / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D002244:Carbon; D011433:Propranolol; D009952:Ornithine