Hydrophobic substances induce water stress in microbial cells

Abstract

Ubiquitous noxious hydrophobic substances, such as hydrocarbons, pesticides and diverse industrial chemicals, stress biological systems and thereby affect their ability to mediate biosphere functions like element and energy cycling vital to biosphere health. Such chemically diverse compounds may have distinct toxic activities for cellular systems; they may also share a common mechanism of stress induction mediated by their hydrophobicity. We hypothesized that the stressful effects of, and cellular adaptations to, hydrophobic stressors operate at the level of water : macromolecule interactions. Here, we present evidence that: (i) hydrocarbons reduce structural interactions within and between cellular macromolecules, (ii) organic compatible solutes - metabolites that protect against osmotic and chaotrope-induced stresses - ameliorate this effect, (iii) toxic hydrophobic substances induce a potent form of water stress in macromolecular and cellular systems, and (iv) the stress mechanism of, and cellular responses to, hydrophobic substances are remarkably similar to those associated with chaotrope-induced water stress. These findings suggest that it may be possible to devise new interventions for microbial processes in both natural environments and industrial reactors to expand microbial tolerance of hydrophobic substances, and hence the biotic windows for such processes.

Figure 1

Activities of hydrophobic substances, and other environmentally relevant stressors, in macromolecular (A) and cellular systems (B) versus log P_{octanol–water} for (A) chaotropic solute activity – quantified using agar gelation as a model system – and (B) inhibitory activity against P. putida. Trend lines are shown in red, and the grey shaded area in (B) indicates the log P region for hydrophobic stressors. The values for chaotropic activity that were calculated from agar : stressor solutions are listed in Table S1; chaotropicity (±1.2 kJ kg⁻¹; see Table S1) and growth‐rate values are means of three independent experiments, and these values were plotted on logarithmic scales.

Figure 2

Inhibition of (A and B) catalytic activity of the model enzyme β‐galactosidase, and (C and D) growth rate of P. putida, by hydrophobic substances and other environmentally relevant stressors: (A) β‐galactosidase activity in the presence of hydrophobic compounds or (B) hydrophilic chaotropes, (C) growth rate of P. putida in media containing hydrophobic compounds or (D) hydrophilic chaotropes. Enzyme assays were carried out independently in duplicate (β‐galactosidase) and P. putida stress tolerance assays were carried out in triplicate; plotted values are means, and standard deviations are shown.

Figure 3

Benzene tolerance of P. putida cells with diverse compatible‐solute contents after growth on media supplemented with glycerol, trehalose or solute stressors (see Table 1) for: (A) cells from high‐trehalose and high‐glycerol media, (B) cells from chaotropic (high‐MgCl₂ and high‐NH₄NO₃) media and (C) cells from low‐water‐activity (high‐glucose or high‐NaCl) media. The stress tolerance assays were carried out in triplicate; plotted values are means and standard deviations are shown.

Figure 4

Diagrammatic illustration of a lipid bilayer showing the locations of hydrophobic substances (e.g. benzene) and hydrophilic chaotropes (e.g. ethanol) that destabilize the structure of lipid bilayers, and the way in which compatible solutes (e.g. betaine) protect against this activity: (A) no added substance (unstressed membrane), (B) benzene‐stressed membrane, (C) ethanol‐stressed membrane, (D) membrane exposed to benzene and betaine and (E) membrane exposed to ethanol and betaine.

Figure 5

Protection of stressor‐inhibited enzymes by diverse compatible solutes for: (A and B) a benzene‐inhibited (A) and a hexane‐inhibited (B) hexokinase‐pyruvate kinase‐lactate dehydrogenase reaction and (C and D) ethanol‐inhibited (C) and MgCl₂‐inhibited (D) β‐galactosidase. All stressors were used to cause 60–90% inhibition of catalytic activity at the following concentrations: benzene 20.5 mM, hexane 123 µM, ethanol 5.2 M and MgCl₂ 0.97 M (see A–D). Compatible‐solute concentrations were: trehalose 5 mM, mannitol 300 mM, glycerol 2000 mM, betaine 1500 mM and proline 78 mM (A and B) and trehalose 62.5 mM, mannitol 75 mM, glycerol 150 mM, betaine 125 mM, proline 156 mM (C and D). All enzyme assays were carried out independently in triplicate (hexokinase assay) or duplicate (β‐galactosidase) and standard deviations are shown.

Figure 6

Intracellular compatible‐solute contents of exponentially growing cells of P. putida (A and B) in minimal mineral‐salt media (at 30°C; see Fig. 2C) supplemented with (A) toluene and (B) 2,5‐dichlorophenol; and those of A. penicillioides (C and D) on MYPiA+sucrose (1.64 M, at 15°C; see Fig. S2) medium supplemented with (C) benzene and (D) octanol over a range of concentrations. Compatible solutes were (◊) glycerol, (▵) mannitol and () trehalose. Plotted values are means of triplicate experiments, and standard deviations are shown.