<FONT FACE="MS Sans Serif" SIZE=2>The aim of the research presented in this thesis was to study the molecular mechanisms of organic solvent tolerance in Pseudomonas putida S12. This bacterium is capable of growth at saturated solvent concentrations, which are lethal to normal bacteria. Organic solvent-tolerant bacteria have potential advantages in either the remediation of highly polluted waste streams or biocatalytic applications for the production of specialty chemicals. The use of these bacteria in biocatalysis would allow the introduction of an organic phase to dissolve water-insoluble substrates or to remove toxic products. As a first step in the identification of genes involved in solvent tolerance, toluene-sensitive transposon mutants of P. putida S12 were generated. As described in Chapter 3, we were able to isolate the genes involved in toluene efflux using the toluene-sensitive strain P. putida JK1. The deduced amino acid sequences encoded by the srpABC genes isolated were highly homologous to proteins involved in proton-dependent efflux. Transfer of the genes for the toluene efflux pump to a normally toluene-sensitive P. putida strain resulted in the acquisition of toluene tolerance. From these results we conclude that organic solvent efflux is the key factor in solvent tolerance. In Chapter 4 it was found that the induction of the membrane associated efflux system SrpABC of P. putida S12 is inducible. Using a reporter vector, containing the srp promoter, it was determined that aromatic and aliphatic solvents and alcohols were capable of inducing the transcription the srpABC genes. However, antibiotics, heavy metals and general stress conditions (pH, temperature, NaCl, and organic acids) did not induce srp transcription. From the results presented in Chapter 4 we conclude that SrpABC-mediated efflux of organic solvents is solely induced by solvent stress. The high levels of antibiotic resistance of P. putida S12 and the relationship between solvent tolerance and antibiotic resistance triggered us to study multidrug resistance in this strain. In analogy to the results presented in Chapter 3 the first step in the identification of genes involved in multidrug resistance was to generate transposon mutants of P. putida S12. In Chapter 5 we describe the isolation the arp genes involved in chloramphenicol efflux, using the isolated chloramphenicol-sensitive P. putida strains CM1 and CM2. Moreover, the ArpABC efflux system was involved in the resistance towards tetracycline, carbenicillin, streptomycin, erythromycin, and novobiocin. Surprisingly, the deduced amino acid sequences encoded by the isolated arpABC genes were highly homologous to proteins involved in proton-dependent efflux of organic solvents. By constructing an arp-srp double mutant it was concluded that arpABC was not involved in efflux of organic solvents. In Chapter 6 octanol-sensitive mutants of P. putida S12 were isolated, which were interrupted in genes for the flagella biosynthetic pathway. These mutants were nonmotile and the formation of the flagellum was totally impaired. The expression of the SrpABC efflux pump in the nonmotile mutants was decreased, possibly due to general regulatory mechanisms. Several genes involved in multidrug resistance and solvent tolerance in P. putida S12 have been isolated and characterized. It would now be interesting to investigate the complex regulation of these systems and to identify new genes using the mutants described in this thesis.
|Qualification||Doctor of Philosophy|
|Award date||13 Mar 2002|
|Place of Publication||S.l.|
|Publication status||Published - 2002|
- industrial microbiology
- pseudomonas putida