Hydrophobic organic solvents, like benzene and toluene, are mainly toxic for bacteria because of their accumulation in the membranes of the cells. The accumulation of solvent molecules in a membrane lowers its rigidity and increases its fluidity and permeability, resulting in an increased rate of cell lysis. Moreover, the functioning of proteins and enzymes embedded in the membranes is negatively affected. In addition, accumulation of solvents causes dissipation of the proton motive force (PMF) as well as a decrease in the energy status of the cell. In 1990 the isolation of the remarkably solvent tolerant bacterium Pseudomonas putida S12 was reported. Research into the solvent tolerance properties of P. putida S12 initially focused on the fatty acid composition of the membranes and how it is influenced by the cell. Later, it was discovered that P. putida S12 is able to actively extrude solvent molecules from the cell by an energy-dependent efflux system that was named Srp, for solvent resistance pump. Furthermore, an insertion sequence named ISS12 was found to play a role in solvent tolerance and a relationship between flagella and solvent tolerance was established. All of the above mentioned mechanisms were elucidated by a traditional reductionist approach. This approach does not provide insight into specific nor global cellular responses related to solvent stress and the interactive dynamics of solvent tolerance mechanisms upon solvent exposure. Systems-level analysis techniques such as transcriptomics and proteomics are very promising tools for elucidating these aspects of solvent tolerance mechanisms. A 2D-DIGE proteomics study of S12 cultured in the presence of 3 mM (sub-lethal) and 5 mM (lethal to non-solvent tolerant bacteria) toluene in chemostats showed that proteins involved in the energy metabolism of the cell play an important role in solvent tolerance (Chapter 2). Five enzymes that are part of the citric acid cycle appeared to be more abundant in toluene-exposed cells than in non-exposed cells. Moreover, other energy-household related proteins showed differential abundances, according to the theory of a higher need for energy in solvent-exposed cells. Among those was AtpF, which is part of the ATP synthase complex, which had a lower abundance in the presence of toluene. Other interesting findings comprised two outer membrane proteins, OprF and OprH, of which the latter one showed the most dramatic increase in abundance. OprH was hypothesised to have a function in positive support of membrane stabilisation. A hypothetical protein, PP3611, appeared to be less abundant in the presence of toluene. The expression of the corresponding gene PP3611 was accordingly shown to be decreased in the presence of toluene as well, in a subsequent transcriptomics experiment (Chapter 3). The gene was renamed trgI (toluene repressed gene I) and knock-out and over-expression mutants were constructed. Analysis of these trgI mutants confirmed the correlation of this gene with solvent tolerance. qPCR analysis led to the hypothesis that trgI plays an important role in the first defence against solvents since its expression decreased immediately after addition of toluene. The knock-out mutant S12?TrgI added to this hypothesis with its increased survival frequency after a 1% toluene shock. The mutant also seemed to exhibit improved lysis resistance as well as a rounded cell morphology and an altered resistance to antibiotics after addition of toluene, suggesting an additional membrane-related function for TrgI. Other results of the transcriptomics analysis of S12 cultured in the presence of toluene comprised differential expression of genes involved in cellular energy-household, as well as genes relating to membrane-associated functions and the outer cell structure. As expected, srpABCRS, encoding the solvent efflux pump and its regulatory genes, was up-regulated. In addition, several flagella- and pili-associated genes were differentially expressed. The function of trgI was further investigated by sudden exposure of S12 and S12?TrgI to 5 mM toluene in batch cultures, and following global expression between 1 and 30 minutes after addition of toluene (Chapter 4). The global transcriptome response revealed large differences between wildtype and trgI deletion mutant. The timing of the overall transcriptional response was delayed in P. putida S12?TrgI. Genes belonging to specific functional groups, i.e. energy production and conversion, amino acid transport and metabolism, lipid metabolism and posttranslational modification, protein turnover and chaperones, some of which have an established relationship with solvent tolerance, were not overrepresented in the trgI deletion mutant after sudden toluene exposure. Specific groups of genes overrepresented amongst the trgI-dependent toluene responsive genes were the same as the groups mentioned above, except for posttranslational modification, protein turnover and chaperones. Moreover, the genes belonging to these groups were consistently overexpressed in the trgI deletion mutant. Hence, in the presence of solvent trgI affected a large group of genes with a large diversity in functions. Analysis of the presumed tertiary structure of the protein TrgI suggested an involvement in regulation of gene expression, which in the light of the broad effects of deletion of the corresponding gene, appears very likely. An improved benzene-tolerant mutant of P. putida S12 was obtained by using laboratory evolution (Chapter 5). S12 was cultured in increasing concentrations of benzene in LB medium for a period of two months, resulting in strain P. putida S12.49 that tolerates up to 24 mM benzene (also see Chapter 6). This strain was cultured in chemostats with and without benzene and global gene expression and protein abundances were compared to those of the wildtype S12 strain. The solvent efflux pump SrpABC was constitutively expressed in S12.49, which appeared to be caused by the insertion of the transposable element ISS12 in the regulatory gene srpS. Since SrpABC is energy-dependent, constitutive expression together with the dissipation of the proton motive force caused by accumulation of benzene molecules in the cell membranes, should hypothetically lead to enhanced expression of genes and proteins related to energy metabolism. Unexpectedly, an overall downregulation of terminal cytochrome c oxidases was observed in S12.49. The additional downregulation of genes involved in the arginine deiminase pathway and the fact that S12.49 still has the wildtype level of benzene tolerance after addition of the energy-uncoupling protonophore CCCP further supported the proposition that S12.49 harbours alternative mechanisms to generate and maintain a proton gradient, or is dramatically more efficient in doing so than the wild-type strain. The research presented in this thesis clearly showed that indeed systems-level analysis techniques such as transcriptomics and proteomics are very useful in identifying the global cellular responses of P. putida S12 to toxic organic solvents (Chapter 6). The thus identified genes can be further investigated using a more classic and targeted approach. This was done for trgI that was hypothesised to be involved in the first line of defence against solvents. Analysis of a knock-out mutant of this gene showed its large influence on gene expression in the presence of solvents. The possible function of the corresponding protein was therefore proposed to be that of a regulator of gene expression. This regulatory function is likely to be influenced by organic solvents. The solvent efflux pump SrpABC has long been considered to constitute the most important mechanism of solvent tolerance in S12. In this thesis it is clearly shown that the intrinsic flexibility of the energy generating mechanisms is at least as important. Without this flexibility, S12 would not be able to make full use of the efflux pump. The combination and interplay of the features that make P. putida S12 solvent tolerant would not have been revealed without the use of transcriptomics and proteomics analyses.