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.

Fig 1. Growth curves of P. putida strains.

(A) all three P. putida DOT-T1E strains in LB medium without propranolol; (B) P. putida DOT-T1E, (C) P. putida DOT-T1E-PS28, and (D) P. putida DOT-T1E-18 in the presence of propranolol. A 1/10 dilution of 100 μL samples were prepared for OD measurement at 660 nm.

Fig 2. PC-DFA scores plots of FT-IR data for three different strains of P. putida strains upon propranolol shock.

Symbols represent different strains. (A) P. putida DOT-T1E is the wild type (stars) and ten PCs with a total explained variance (TEV) of 99.43% were used for the DFA, (B) P. putida DOT-T1E-PS28 is the mutant (closed triangles) and ten PCs with a TEV of 99.65% were used for the DFA, (C) P. putida DOT-T1E-18 is the mutant (closed circles) and ten PCs with a TEV of 99.03% were used for the DFA. Colour coding: control with no propranolol (red), cells exposed to 0.2 mg mL^-1 propranolol (black), 0.4 mg mL^-1 propranolol (brown), and 0.6 mg mL^-1 propranolol (blue). Arrows indicate the direction of shift because of the increase of propranolol concentration. (D) PC-DFA loadings plot for P. putida DOT-T1E. (E) PC-DFA loadings plot for P. putida DOT-T1E-PS28, (F) PC-DFA loadings plot for P. putida DOT-T1E-18. Significant loadings were assigned to bacterial proteins.

Fig 3. Box-whisker plots showing the changes in metabolite levels in control and cells exposed to propranolol for 4 biological replicates.

Variable 180 was identified as propranolol. (Red line) indicates the median m/z intensity. (A) Represent the data for 3 P. putida strains, 4 concentrations of propranolol and 3 time points, dashed lines separate different concentration levels of propranolol and solid line separates different strains. (B) Represent the data for 3 P. putida strains, 3 concentrations of propranolol and 1 time point at 60 min, dashed lines separates different strains.

Fig 4. Box-whisker plot showing the changes in ornithine levels (variable id 100) in control and exposed cells to propranolol.

(Red line) indicates the median m/z intensity. These plots represent the data for 3 P. putida strains, 4 concentrations of propranolol and 3 time points, for 4 biological replicates. Dashed lines separate different concentration levels of propranolol and solid line separates different strains.

Fig 5. Schematic metabolic diagram of central carbon metabolism in P. putida DOT-T1E adapted to propranolol showing the level of metabolites for cells exposed to propranolol compared to the control.

Metabolites were detected and identified by GC-MS. Metabolites indicated in black were observed, while metabolites indicated in grey were not detected. The median m/z intensity (red line) in the box- whisker plots was used to compare the level of metabolites. (A) Represent the level of metabolites at 10 min, while (B) the level of metabolite at 60 min. Traffic light system represents different concentration of propranolol. Red, yellow and green represent exposed cells to 0.2, 0.4 and 0.6 mg/mL of propranolol respectively. Up-arrow, down-arrow and steady arrow indicate an increase, a decrease and no change in the level of metabolite respectively. The number of arrows represents the level of metabolites. Slight change (single arrow), medium change (double arrows) and high change (triple arrows).

Fig 6. Schematic diagram of central carbon metabolism in P. putida DOT-T1E-18 adapted to propranolol showing the level of metabolites for cells exposed to propranolol compared to the control ones.

Metabolites were detected and identified by GC-MS. Metabolites indicated in black were observed, while metabolites indicated in grey were not detected. The median m/z intensity (red line) in the box- whisker plots was used to compare the level of metabolites. (A) Represent the level of metabolites at 10 min, while (B) the level of metabolite at 60 min. Traffic light system represents different concentration of propranolol. Red, yellow and green represent exposed cells to 0.2, 0.4 and 0.6 mg/mL of propranolol respectively. Up-arrow, down-arrow and steady arrow indicate an increase, a decrease and no change in the level of metabolite respectively. The number of arrows represents the level of metabolites. Slight change (single arrow), medium change (double arrows) and high change (triple arrows).