S. Isken

Bacteria tolerant to organic solvents.

Abstract 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 Pow). Substances with a log Pow 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.

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

Defence mechanisms of three Pseudomonas putida strains growing in the presence of toluene up to 50% (v/v) were investigated. The three strains reacted to toxic concentrations of toluene by accumulating trans unsaturated fatty acids in the membrane instead of the cis isomers. The membranes of the toluene-adapted cells possessed a higher trans/cis ratio and had a higher lipid-ordering since the transition temperature was about 7 centigrade degrees higher compared to the non-adapted cells.

Ethanol tolerance and membrane fatty acid adaptation in adh multiple and null mutants of Kluyveromyces lactis

The effects of ethanol and 1-octanol on growth and fatty acid composition of different strains of Kluyveromyces lactis containing a mutation in the four different alcohol dehydrogenase (KlADH) genes were investigated. In the presence of ethanol and 1-octanol K. lactis reduced the fluidity of its lipids by decreasing the unsaturation index (UI) of its membrane fatty acids. In this way, a direct correlation between nonlethal ethanol concentrations and the decrease in the UI could be observed. At concentrations which totally inhibited cell growth no reaction occurred. These adaptive modifications of the fatty acid pattern of K. lactis to ethanol contrasted with those reported for Saccharomyces cerevisiae and Schizosaccharomyces pombe. Whereas these two yeasts increased the fluidity of their membrane lipids in the presence of ethanol, K. lactis reduced the fluidity (UI) of its lipids. Among the different isogenic adh negative strains tested, the strain containing no ADH (adh0) and that containing only KlADH1 were the most alcohol-sensitive. The strain with only KlADH2 showed nearly the same tolerance as reference strain CBS 2359/152 containing all four ADH genes. This suggests that the KlADH2 product could play an important role in the adaptation/detoxification reactions of K. lactis to high ethanol concentrations.

Effect of solvent adaptation on the antibiotic resistance in Pseudomonas putida S12

Abstract The effect of the adaptation to toluene on the␣resistance to different antibiotics was investigated in the␣solvent-resistant strain Pseudomonas putida S12. We␣followed the process of the solvent adaptation of P.␣putida S12 by cultivating the strain in the presence␣of␣increasing concentrations of toluene and studied␣the correlation of this gradual adaptation to the resistance towards antibiotics. It was shown that the tolerance to various chemically and structurally unrelated antibiotics, with different targets in the cell, increased during this gradual adaptation. The survival of P. putida S12 in the presence of antibiotics like tetracycline, nigericin, polymyxin B, piperacillin or chloramphenicol increased 30- to and 1000-fold after adaptation to 600 mg/l toluene. However, cells grown in the absence of any solvents lost their adaptation to toluene even when grown in the presence of antibiotics. Results are discussed in terms of the physico-chemical properties of membranes as affected by the observed cis/trans isomerization of unsaturated fatty acids, as well as in terms of the active efflux of molecules from the cytoplasmic membrane.

Isolation and characterization of the solvent-tolerant Bacillus cereus strain R1.

Toluene-tolerant gram-positive bacteria were isolated and identified to belong to the genus Bacillus. They grew in a medium containing yeast extract and in the presence of a separate phase of toluene or other hydrocarbons, but not when aliphatic alcohols were present. The isolate Bacillus cereus R1 did not metabolise or transform toluene. Toluene accumulation in its cells was rapid, unless the organism was supplied with glucose as energy source. In bacteria adapted to toluene, the amount of toluene accumulating in cells was one-half that in nonadapted bacteria. Valinomycin (K+ ionophore) and o-vanadate (ATPase inhibitor) as inhibitors of energy metabolism partly counteracted the effect of glucose as energy source. These results suggest the presence of an efflux mechanism for toluene in strain R1. The nature of this mechanism and its function in a solvent-tolerant gram-positive strain are discussed.

The solvent efflux system of Pseudomonas putida S12 is not involved in antibiotic resistance

Abstract The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [14C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [14C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps.