Physiology of solvent tolerance in Pseudomonas putida S12

Abstract

Hydrophobic organic solvents, like toluene, are toxic for living organisms. This toxicity is an important drawback in the environmental biotechnology as well as in the application of solvents in the production of fine chemicals by whole-cell biotransformations. The effects of organic solvents on micro-organisms have been studied extensively. It was shown that the toxicity of hydrophobic organic solvents is mainly caused by the ability of such solvents to intercalate and accumulate in biological membranes.In the last decade, however, several strains that can survive the presence of toxic organic solvents have been isolated. One of the solvent-tolerant strains is Pseudomonas putida S12, studied in this thesis. This strain can grow in the presence of a second phase of organic solvents that have a log P O/W (logarithm of the partition coefficient between octanol and water) value equal to or higher than 2.3. P. putida S12 is able to survive the presence of solvents because of different adaptation mechanisms. This strain can suppress the effect of organic solvents on the membranes by a cis to trans isomerization of the unsaturated fatty acids of the membrane.A further adaptation mechanism is presented in this thesis. It is shown that cells adapted to toluene posses an active export system for toluene. Therefore, P. putida S12 compensates not only the toxic effects of organic solvents on the membrane, but decrease also actively the amount of the toxic solvent in the cell. In the presence of the efflux system the concentration of a solvent in the bacterial membrane can be below the theoretical equilibrium. This active efflux system depends on the proton motive force.Since we reported the presence of an active export system for solvents, it has been suggested repeatedly, that this system is connected with the well-described efflux systems for antibiotics. Indeed, the adaptation of P. putida S12 to toluene enhances the resistance of this strain to various chemically and structurally unrelated antibiotics, with different targets in the cell.However, we could demonstrate that efflux system for toluene in P. putida S12 does not export antibiotics. This efflux system is specific for solvents like toluene and p -xylene. Therefore, the adaptation to an organic solvent must activate other mechanisms responsible for the resistance towards these antibiotics. Likely, this is connected to a general stress response. This general stress response may also cause the decrease of the cell-envelope permeability discussed in the thesis.The broad effect solvents have even on the solvent-tolerant strain P. putida S12 are demonstrated. The presence of toluene reduces the maximum growth yield and increases the maintenance requirement. Interestingly, other solvents had a similar effect as toluene as long as they reached the same concentration in the bacterial membrane. Not the chemical structure but the amount of solvent accumulated in the bacterial membrane determines the effect of a solvent on the cells. Therefore, results obtained with toluene can be extrapolated to other solvents as well.</p