Metabolomic investigation of the bacterial response to a metal challenge

Abstract

Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a "poised" physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.

FIG. 1.

Growth curves of P. pseudoalcaligenes KF707 wild-type and T5 strains in the presence of K₂TeO₃ (25 μg ml⁻¹, ∼100 μM) in LB and MSM with different carbon sources. Growth of KF707 in succinate and fumarate shows a lag phase of 10 to 16 h (B and C), comparable to the lag phase for growth in LB (A). With malate, growth does not require a phase of adaptation (D). In contrast to that of wild-type KF707, growth of T5 is characterized by the lack of initial lag phase and increased MICs under all conditions tested in this study. Representative growth curves obtained in one experiment are shown here. (A) LB; (B) MSM-succinate (0.3%, wt/vol); (C) MSM-fumarate (0.3%, wt/vol); (D) MSM-malate (0.3%, wt/vol).

FIG. 2.

Effects of tellurite on viability and RSH loss of wild-type and T5 cells exposed to K₂TeO₃ (25 μg ml⁻¹) during exponential growth in LB. Viability is expressed as a percentage of viable cells recovered after 30 or 60 min of exposure compared to the initial number of viable cells (5% standard deviation). RSH loss is expressed as a percentage of oxidized RSH at a given time point during exposure to tellurite over the initial content (5 to 8% standard deviation). Negative values indicate RSH oxidation.

FIG. 3.

Membrane potential measurements for KF707 wild-type (left) and T5 (right) cells. The values of the membrane potentials measured for the wild-type and T5 strains in the presence (+TeO₃²⁻) or absence (LB) of tellurite are indicated at the far right. The dashed traces show the distribution of the TPP⁺ cation (1 μM) after the addition of tellurite to KF707 (left) and T5 (right) cells growing in LB. VAL, valinomycin (4 μM); NIG, nigericin (3 μM); FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 μM).

FIG. 4.

Representative ¹H NMR spectra from each of the four samples tested in the metabolomics analysis (i.e., wild-type KF707, T5, KF707 with tellurite, and T5 with tellurite). The insets display a zoomed region of the spectra corresponding to the metabolite glycerol, shown as an example of significant variation across the samples and contributing to the multivariate models (see Fig. 5).

FIG. 5.

Coefficient plots showing the increase or decrease in concentration for each of the metabolites identified by targeted profiling. Each plot corresponds to the OPLS-DA model in Table 1, comparing the wild type with T5 (A), the wild type with and without tellurite (B), and T5 with and without tellurite (C). The coefficient along the y axis is a measure of both the magnitude and direction of change of metabolite concentrations between the two states compared in each model. The black dots indicate which metabolites contribute significantly to the OPLS-DA models (VIP of >1; see Materials and Methods for further details). Error bars indicate standard deviations.

FIG. 6.

VIP-SUS plot for the models comparing the wild type with and without tellurite and T5 with and without tellurite. Metabolites along the x axis are important for the wild-type model (lower right quadrant), whereas metabolites along the y axis are important for the T5 model (upper left quadrant). Metabolites with x and y values higher than 1 are important for both models (upper right quadrant). A variable is considered important to a model if the VIP is >1 (see Materials and Methods for further details). The VIP value is a composite measure of a metabolite's importance in explaining the statistical variance and the predictive value of the model.