Mechanisms of membrane toxicity of hydrocarbons.

The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than their natural aqueous reaction media. Studies over the past 15 years have revealed not only that this change in solvent is feasible, but also that in such seemingly hostile environments enzymes can catalyse reactions impossible in water, become more stable, and exhibit new behaviour such as 'molecular memory'. Of particular importance has been the discovery that enzymatic selectivity, including substrate, stereo-, regio- and chemoselectivity, can be markedly affected, and sometimes even inverted, by the solvent. Enzyme-catalysed reactions in organic solvents, and even in supercritical fluids and the gas phase, have found numerous potential applications, some of which are already commercialized.

Improving enzymes by using them in organic solvents

Each recent report of liquid water existing elsewhere in the Solar System has reverberated through the international press and excited the imagination of humankind. Why? Because in the past few decades we have come to realize that where there is liquid water on Earth, virtually no matter what the physical conditions, there is life. What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harbouring 'extremophiles'. This realization, coupled with new data on the survival of microbes in the space environment and modelling of the potential for transfer of life between celestial bodies, suggests that life could be more common than previously thought. Here we examine critically what it means to be an extremophile, and the implications of this for evolution, biotechnology and especially the search for life in the Universe.

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Life in Extreme Environments

The natural antimicrobial compound carvacrol shows a high preference for hydrophobic phases. The partition coefficients of carvacrol in both octanol-water and liposome-buffer phases were determined (3.64 and 3.26, respectively). Addition of carvacrol to a liposomal suspension resulted in an expansion of the liposomal membrane. Maximum expansion was observed after the addition of 0.50 μmol of carvacrol/mg of l-α-phosphatidylethanolamine. Cymene, a biological precursor of carvacrol which lacks a hydroxyl group, was found to have a higher preference for liposomal membranes, thereby causing more expansion. The effect of cymene on the membrane potential was less pronounced than the effect of carvacrol. The pH gradient and ATP pools were not affected by cymene. Measurement of the antimicrobial activities of compounds similar to carvacrol (e.g., thymol, cymene, menthol, and carvacrol methyl ester) showed that the hydroxyl group of this compound and the presence of a system of delocalized electrons are important for the antimicrobial activity of carvacrol. Based on this study, we hypothesize that carvacrol destabilizes the cytoplasmic membrane and, in addition, acts as a proton exchanger, thereby reducing the pH gradient across the cytoplasmic membrane. The resulting collapse of the proton motive force and depletion of the ATP pool eventually lead to cell death.

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The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus

Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times ( 2 S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.

Recent Advances in Petroleum Microbiology

The toxic effects that organic solvents have on whole cells is an important drawback in the application of these solvents in environmental biotechnology and in the production of fine chemicals by whole-cell biotransformations. Hydrophobic organic solvents, such as toluene, are toxic for living organisms because they accumulate in and disrupt cell membranes. The toxicity of a compound correlates with the logarithm of its partition coefficient with octanol and water (log P(ow)). Substances with a log P(ow) value between 1 and 5 are, in general, toxic for whole cells. However, in recent years different bacterial strains have been isolated and characterized that can adapt to the presence of organic solvents. These strains grow in the presence of a second phase of solvents previously believed to be lethal. Different mechanisms contributing to the solvent tolerance of these strains have been found. Alterations in the composition of the cytoplasmic and outer membrane have been described. These adaptations suppress the effects of the solvents on the membrane stability or limit the rate of diffusion into the membrane. Furthermore, changes in the rate of the biosynthesis of the phospholipids were reported to accelerate repair processes. In addition to these adaptation mechanisms compensating the toxic effect of the organic solvents, mechanisms do exist that actively decrease the amount of the toxic solvent in the cells. An efflux system actively decreasing the amount of solvents in the cell has been described recently. We review here the current knowledge about exceptional strains that can grow in the presence of toxic solvents and the mechanisms responsible for their survival.

Bacteria tolerant to organic solvents

Solvent-tolerant bacteria in biocatalysis

Defence mechanisms of three Pseudomonas putida strains growing in the presence of toluene up to 50%

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Cis/trans isomerization of fatty acids as a defence mechanism of Pseudomonas putida strains to toxic concentrations of toluene

Nitroaromatic compounds are abundantly present in nature, but are in most cases highly toxic to living organisms. Several microorganisms, however, are capable of mineralizing or converting these compounds. Until now four pathways for the complete degradation of nitroaromatics have been described, which start with either the oxygenolytic or reductive removal of the nitro group from the aromatic ring or with this removal by means of replacement reactions. Besides these conversions many organisms are able to reduce nitroaromatics. The degradation of nitroaromatic compounds does not only occur in pure cultures but also in situ, for example in soil, water and sewage. However, several problems are associated with the application of microorganisms in the bioremediation of contaminated sites, as nitroaromatics or their conversion products may chemically interact with soil particles and cells. Besides the possibilities of applying microorganisms in the cleaning of sites contaminated with nitroaromatics, the use of microorganisms or enzymes in the biocatalytic production of industrially valuable products from nitroaromatics is also discussed.

Degradation of nitroaromatic compounds by microorganisms.

Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this nl ea. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in sevaral instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur: ln addition to the effects of lipophilic compounds on the lipid part of the membrane proteins embedded in the membrane ale affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering change in lipid/protein ratio) that compensate for the changes in membrane structure are treated.

J.A.M. de Bont

Papers

Bacteria tolerant to organic solvents

Solvent-tolerant bacteria in biocatalysis

Cis/trans isomerization of fatty acids as a defence mechanism of Pseudomonas putida strains to toxic concentrations of toluene

Degradation of nitroaromatic compounds by microorganisms.

Mechanisms of membrane toxcity of hydrocarbons.