title: Metabolic analysis of the response ofPseudomonas putidaDOT-T1E strains to toluene using Fourier transform infrared spectroscopy and gas chromatography mass spectrometry.
abstract: BACKGROUND
  An exceptionally interesting stress response ofPseudomonas putidastrains to toxic substances is the induction of efflux pumps that remove toxic chemical substances from the bacterial cell out to the external environment. To exploit these microorganisms to their full potential a deeper understanding of the interactions between the bacteria and organic solvents is required. Thus, this study focuses on investigation of metabolic changes inP. putidaupon exposure to toluene.
  
  
  OBJECTIVE
  Investigate observable metabolic alterations during interactions of three strains ofP. putida(DOT-T1E, and its mutants DOT-T1E-PS28 and DOT-T1E-18) with the aromatic hydrocarbon toluene.
  
  
  METHODS
  The growth profiles were measured by taking optical density (OD) measurement at 660 nm (OD660) at various time points during incubation. For fingerprinting analysis, Fourier-transform infrared (FT-IR) spectroscopy was used to investigate any phenotypic changes resulting from exposure to toluene. Metabolic profiling analysis was performed using gas chromatography-mass spectrometry (GC-MS). Principal component-discriminant function analysis (PC-DFA) was applied to the FT-IR data while multiblock principal component analysis (MB-PCA) andN-way analysis of variance (N-way ANOVA) were applied to the GC-MS data.
  
  
  RESULTS
  The growth profiles demonstrated the effect of toluene on bacterial cultures and the results suggest that the mutantP. putidaDOT-T1E-18 was more sensitive (significantly affected) to toluene compared to the other two strains. PC-DFA on FT-IR data demonstrated the differentiation between different conditions of toluene on bacterial cells, which indicated phenotypic changes associated with the presence of the solvent within the cell. Fifteen metabolites associated with this phenotypic change, inP. putidadue to exposure to solvent, were from central metabolic pathways. Investigation of MB-PCA loading plots andN-way ANOVA for condition | strain × time blocking (dosage of toluene) suggested ornithine as the most significant compound that increased upon solvent exposure.
  
  
  CONCLUSIONS
  The combination of metabolic fingerprinting and profiling with suitable multivariate analysis revealed some interesting leads for understanding the mechanism ofPseudomonasstrains response to organic solvent exposure.
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Ali
authors.identifier: 0000-0002-9393-6347
authors.initials: A
authors.lastname: Sayqal
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xu
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Drupad K
authors.identifier: 0000-0001-7969-0195
authors.initials: DK
authors.lastname: Trivedi
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Najla
authors.initials: N
authors.lastname: AlMasoud
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: David I
authors.identifier: 0000-0002-7633-7019
authors.initials: DI
authors.lastname: Ellis
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Howbeer
authors.initials: H
authors.lastname: Muhamadali
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Nicholas J W
authors.initials: NJ
authors.lastname: Rattray
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Carole
authors.initials: C
authors.lastname: Webb
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN UK.
authors.forename: Royston
authors.identifier: 0000-0003-2230-645X
authors.initials: R
authors.lastname: Goodacre
keywords: Efflux pumps; FT-IR; GC–MS; Metabolomics; Ornithine; P. putida DOT-T1E; Tolerance; Toluene