3.3. FT-IR Fingerprinting of P. putida DOT-T1E Cultures
In recent years, much attention in the literature has been paid to investigating stress responses in bacteria via the application of metabolomics-based methods [
54,
55,
56], an area which has been applied to a broad range of disciplines including medical sciences, metabolic engineering and drug discovery [
57,
58,
59,
60].
In this study, a metabolic fingerprinting approach [
33] based on FT-IR spectroscopy [
61] was employed to study the influence of metal ions on the whole-organism phenotype of
P. putida DOT-T1E strains in the presence and absence of toluene. To ensure that there was sufficient biomass for metabolomics analysis, cultures were grown in LB medium supplemented without or with 7 mM magnesium and 3 mM calcium in the absence/presence of 0.05% (
v/
v) toluene and incubated for 8 h (see
Figure 2). The results showed that all strains have the ability to grow in the absence/presence of toluene; however, all
P. putida strains had higher tolerance to toluene in the presence of divalent cations. By contrast, similar growth profiles were observed in the culture medium supplemented with or without divalent cations in the absence of toluene.
To generate robust biochemical fingerprints of
P. putida DOT-T1E strains, FT-IR spectroscopy was employed. Since subtle and important variations in FT-IR spectra are not easy to interpret visually (
Figure S4), chemometric methods were conducted in order to analyse these data in far more detail. Initially, a PCA scores plot was produced (data not shown) and no obvious clusters were observed in this analysis. PCA failed to discriminate data as in many previous studies [
44,
62]. Therefore, it would seem sensible to employ a supervised clustering approach such as DFA in order to visualise the distribution of samples based on their IR metabolic fingerprint [
34]. The first and second discriminant function (DF) scores were generated to identify variation or relationships between the samples, and the resultant PC-DFA scores plot of DF1
vs. DF2 is displayed in (
Figure 3). As can be seen in
Figure 3, a clear separation between the wild-type DOT-T1E and the mutants DOT-T1E-PS28 and DOT-T1E-18 is observed in the first discriminant function which explains the majority of the total group variance (here the groups relate to the biological replicates and are not biased based on either the level of toluene or the addition of cations). This observation could be due to the lack of efflux pump in the mutants compared to the parent strain or an indirect effect on growth of mutant DOT-T1E-18, indicating the ability of FT-IR to discriminate between bacterial cells within the same strain.
Figure 3 also clearly shows that a similar trend (through DF2) was observed between the wild-type and the mutants under the same conditions, indicating clear metabolic changes caused by metal ions in the absence and presence of toluene. The parent and the mutant strains have the same genetic background and the only difference between the three cell types is the absence of one of the efflux pump proteins in the mutants compared with the parent strain. Therefore, the results from DFA would suggest that the influence of Mg
2+ and Ca
2+ on the stabilization of the toluene tolerance of
P. putida DOT-T1E may be due to the contribution of metal ions in other bacterial-tolerance mechanisms rather than only the efflux pump(s).
In addition, cells exposed to 0.05% (
v/
v) toluene in the absence of metal ions in the wild-type
P. putida DOT-T1E and the mutant
P. putida DOT-T1E-PS28 (a mutant in the TtgGHI pump) are clustered more closely to the control cultures compared with the mutant
P. putida DOT-T1E-18 (which lacks the TtgABC pump), indicating that DOT-T1E-18 cells were more sensitive to 0.05% (
v/
v) toluene compared to DOT-T1E and DOT-T1E-PS28 cells. This clustering pattern would suggest that the TtgABC pump might play a more crucial role in toluene efflux than the TtgGHI pump. This observation was in agreement with previous investigations which conclude that the TtgABC pump is the main extrusion pump, and is able to extrude solvents and antibiotics [
63,
64,
65]. Therefore, the results from DFA clearly illustrate that the metabolic fingerprinting approach has the ability to detect a clear effect upon the cell cultures caused by metal ions and toluene which may cause changes to the phenotype of cells.
To investigate which spectral regions discriminated between different conditions within strains, DFA loadings vectors were calculated and plotted for DF2 (
Figure 4) which largely discriminated between different conditions (
Figure 3). Several changes occur within these loading plots with the greatest variances being observed between 2950–2850 cm
−1, 1700–1600 cm
−1 and 1110–945 cm
−1 contributed to the DFA score plot clustering. Vibrational assignments are provided in
Table 1; in this region of mid-infrared, the bands at 2918 cm
−1 and 2853 cm
−1 can be attributed to C-H stretching vibrations from membrane lipids and the peaks at 1630 cm
−1 and 1550 cm
−1 would be attributed to C=O stretching (amide I) and a combination of C-N stretching and N-H bending (amide II) vibrations, respectively, from protein components. In addition, the bands at 1105 cm
−1 and 952 cm
−1 could arise from a range of vibrations from the carbohydrates family including complex polysaccharide within the cells. These large variations in lipids, proteins and carbohydrates between different conditions within the
P. putida DOT-T1E cells are due to the biological effects caused by the metal ions and toluene.
The outer membrane of Gram-negative bacteria is an effective barrier for many toxic agents, and divalent cations (in particular, Mg
2+ and Ca
2+) are important in the organisation of the outer membrane [
67] as lipopolysaccharide (LPS) molecules are linked to each other electrostatically via divalent cations [
68,
69]. In several cases it has been observed that when the structure of the outer membrane of certain organisms (which are able to acquire resistance against toxic solvents (e.g., toluene)), are modified by chemical or enzymatic removal of parts of the LPS molecule or mutation, the resistance of these bacteria to these solvents is decreased [
70,
71,
72]. On the other hand, Junker
et al. (2001) observed that in a WbpL mutant of
P. putida DOT-T1E, LPS may not be important for aromatic hydrocarbon tolerance [
73]. If Mg
2+ and Ca
2+ are essential for the integrity of the outer membrane and LPS layer, the presence of many aromatic hydrocarbons (e.g., toluene), ethylenediaminetetraacetic acid (EDTA) and antimicrobial peptides (AMPs), lead to significant changes in the structure and function of membrane components, such as disruption and removal of lipids and proteins as well as loss of Mg
2+ and Ca
2+ [
2,
74,
75,
76].
In Gram-negative bacteria, Clifton
et al. [
77] reported that the removal of calcium ions from the LPS bilayer led to the destabilisation of the bilayer and mixing of LPS molecules between the inner and outer leaflets; indicating the important role of salt bridges which are formed by divalent cations (e.g., Mg
2+ and Ca
2+) with negatively charged sugar in LPS core oligosaccharide to strengthen the integrity of the outer membrane. It has been found that calcium has the ability to block the binding of a cationic antimicrobial peptide to LPS and thus decrease its antimicrobial activity [
78]. The effect of AMPs, EDTA and Mg
2+ on the LPS layer was examined in Gram-negative bacteria [
77], showing that cationic AMPs or anionic EDTA effectively modify the LPS layer electrostatically by displacing Mg
2+ ions from the LPS layer competitively, while Mg
2+ tightens and stabilises the LPS layer [
79].
Therefore, it is perhaps not surprising that a similar trend in the DFA scores plot between the wild-type and the mutants were observed, suggesting the contribution of Mg2+ and Ca2+ in LPS stabilisation but not efflux pumps. This observation would suggest that the efflux pumps system in P. putida might not require a magnesium or calcium gradient to export substrates such as toluene. In addition, the most significant changes observed from the interpretation of FT-IR spectra were in the vibration frequency of the polysaccharide, protein and lipid components, and we can infer from this that the important role of divalent cations in P. putida DOT-T1E strain is related to LPS mechanism to cope with the presence of toluene.
Finally, the ratio of saturated fatty acid composition was calculated from the raw (
Figure 5) and scaled infrared spectra (
Figure S5) to investigate the effect of divalent cations and toluene on
P. putida DOT-T1E strains. It is clear that upon toluene exposure, the saturated fatty acid ratio (CH
3:CH
2) was lower compared to the control cultures in the absence of these divalent cations. This result is in agreement with previous observations showing that the fluidity of
P. putida S12 outer membrane increased in the presence of toluene, as toluene may displace divalent cations from the LPS layer, causing increased membrane permeability [
80]. By contrast, the saturated fatty acid ratio of
P. putida cells was increased with the addition of Mg
2+ and Ca
2+ to medium with and without toluene. However, under the same conditions there was a slight decrease in the saturated fatty acid ratio for
P. putida DOT-T1E-PS28 in the presence of Mg
2+. In
Pseudomonas aeruginosa, Schneck
et al. [
81] were able to show that the conformation of the O-antigen was shorter and had a denser layer in the presence of Ca
2+ compared to the absence of calcium ions. Our results would suggest that divalent cations are essential for the integrity of the LPS layer and the outer membrane and therefore they may play an important role to improve solvent tolerance in
P. putida cells.