Mechanisms for Solvent Tolerance in Pseudomonas putida S 12
Sonja ISKEN, Jasper KIEBOOM, and Jan A.M. De BONT*
Division of Industrial Microbiology, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands
In 1989 Inoue and Horikoshi (1) reported the isolation of a toluene-tolerant Pseudomonas putida strain able to grow in a two-phase toluene-water system. This finding came as a surprise because toluene is extremely toxic for living organisms. It accumulates in and disrupts membranes (2). The initial observations by Inoue and Hirokoshi have been confirmed by others (3,4) and we have isolated and described the solvent tolerant Pseudomonas putida S12 (5).
Solvents are toxic to microorganism because they accumulate in the cytoplasmic membrane (6). The numerous observations of toxic effects of solvents on microorganisms can be explained largely by the interaction of these compounds with the membrane. The cytoplasmic membrane looses its integrity as a result of the accumulation of the solvents, and an increase in permeability to protons and ions can be observed (7). These effects on the structural and functional properties of membranes are general in the sense that they are not attributed to a specific reactive group in a molecule.
Mechanisms responsible for the resistance in the newly isolated Pseudomonas putida strains to solvents have been studied. It appears two major systems are in operation. On the one hand, membrane modifications are an important aspect (8) and a key process is the isomerization of cis- into trans-unsaturated fatty acids. Adaptations in the phospholipid headgroups have also been observed. On the other hand, however, such modifications can only be partly successful in combatting the devasting effects of solvents. Sooner or later, the solvent will accumulate to a level which inevitably will destroy the structure of the membrane. It very recently has been assessed an active solvent-efflux system is present in solvent-tolerant bacteria (9). By using 14C-toluene it was found the compound is exported from the membrane in an energy-dependent process. It was the first demonstration of an active transport system for uncharged small molecules over a bacterial membrane. It explains how the solvent-tolerant strains prevent the accumulation of solvents in their cytoplasmic membrane.
The nature of the efflux pump has been uncovered this year. In collaboration with the group of Prof G. Zylstra at Rutgers University in the USA the genes for the solvent efflux pump from P. putida S 12 were cloned and their nucleotide sequences determined (10). The genes involved were labeled srpABC for solvent resistance
