The development of tolerance in Pseudomonas putida DOT-T1 to toluene and related highly toxic compounds involves short-and long-term responses. The short-term response is based on an increase in the rigidity of the cell membrane by rapid transformation of the fatty acid cis-9,10-methylene hexadecanoic acid (C17:cyclopropane) to unsaturated 9-cis-hexadecenoic acid (C16:1,9 cis) and subsequent transformation to the trans isomer. The long-term response involves in addition to the changes in fatty acids, alterations in the level of the phospholipid polar head groups: cardiolipin increases and phosphatidylethanolamine decreases. The two alterations lead to increased cell membrane rigidity and should be regarded as physical mechanisms that prevent solvent penetrance. Biochemical mechanisms that decrease the concentration of toluene in the cell membrane also take place and involve: (i) a solvent exclusion system and (ii) metabolic removal of toluene via oxidation. Mutants unable to carry out cis 3 trans isomerization of unsaturated lipids, that exhibit altered cell envelopes because of the lack of the OprL protein, or that are unable to exclude toluene from cell membranes are hypersensitive to toluene.Organic solvents with a logP OW value (logarithm of the partition coefficient of the target compound in a mixture of octanol/ water) between 1.5 and 3 are extremely toxic to microorganisms, a characteristic that has been well documented for toluene (logP OW 2.5) (1-4). De Smet et al. (2) demonstrated that toluene destabilizes the inner membrane of Gram-negative bacteria, causing a transition from a lamellar bilayer state to a hexagonal state, which results in the leakage of proteins, lipids, and ions and disruption of the cell membrane potential (1, 2). The consequent collapse of ATP synthesis together with other lesions lead to cell death.Inoue and Horikoshi (5) isolated a Pseudomonas putida strain able to grow in a double phase system that contained up to 50% (v/v) toluene, despite the fact that this microorganism was not able to use this aromatic as a carbon source. This report was followed by three independent studies that described the isolation of three different P. putida strains that tolerated related organic solvents, e.g. styrene (6), xylenes (7), and toluene (8). The toluene-tolerant isolate, called P. putida DOT-T1, metabolized toluene via the p-cresol pathway (8). The "unexpected" ability of these Pseudomonas strains to tolerate toxic solvents opens new avenues of research into cellular metabolism. In this study, we have explored the molecular basis for solvent tolerance by P. putida DOT-T1. EXPERIMENTAL PROCEDURESBacterial Strains and Culture Conditions-P. putida DOT-T1 is a solvent-tolerant strain (8), whereas P. putida mt-2 is a toluene-sensitive strain (9).Isolation of Toluene-sensitive Tn5 Mutants of P. putida DOT-T1-About 2000 Tn5 transconjugants of P. putida DOT-T1 were obtained after mating this strain with Escherichia coli (pGS9). The suicide plasmid pGS9 bears Tn5, and mutagenesis was carried out a...

Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source. The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log P ow (octanol/water partition coefficient) was higher than 2.3. Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. Mg 2؉ ions in the culture medium improved solvent tolerance. Electron micrographs showed that cells growing on high concentrations of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity. Polarographic studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolyze toluene. Although the strain also thrived in high concentrations of m-and p-xylene, these hydrocarbons could not be used as the sole C source for growth. The catabolic potential of the isolate was expanded to include m-and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km.Reports of the toxicity of aromatic solvents to microorganisms first appeared early in this century (reviewed in reference 21). Aromatic hydrocarbons become toxic when they are partitioned into lipid bilayer membranes, leading to significant changes in the structure and functioning of membrane components, e.g., disruption of the membrane potential, removal of lipids and proteins, and loss of Mg 2ϩ and Ca 2ϩ cations as well as other small molecules (21). Although a number of microbes able to grow at the expense of aromatic compounds have been isolated, their addition to the culture medium, in general, prevented growth and bacteria were able to survive only when these compounds were supplied in the vapor phase (7,27).There is considerable interest in the isolation of microbes able to thrive in high concentrations of organic solvents, because these microbes can be used as vehicles for the elimination of low-molecular-weight aromatic compounds such as toluene, styrene, benzene, and xylenes, the removal of which is of high priority (14). Furthermore, these aromatic hydrocarbons can be converted into value-added compounds such as cisdiols, epoxides, and indigo, among others (6,7,15,26). Their current synthesis by biological means requires large amounts of water, a major cost in the fermentation industry (10). Therefore, synthesis in double-phase fermentors could be more economical. Lastly, understanding of the mechanisms of solvent tolerance can be exploited in the future to generate microbes with enhanced biocatalytic potential. Inoue et al. (12,13) reported that certain Pseudomonas strains were able to thrive in the presence of more than 50% (vol/vol) toluene, although these strains were not able to grow with the aromatic hydrocarbon alone and other sources of carbon and energy were required. Cruden et al. (3) and Weber et al. (23) reported the isolation of bacterial strains able to grow at th...

The titanocene-catalyzed cascade cyclization of epoxypolyenes, which are easily prepared from commercially available polyprenoids, has proven to be a useful procedure for the synthesis of C(10), C(15), C(20), and C(30) terpenoids, including monocyclic, bicyclic, and tricyclic natural products. Both theoretical and experimental evidence suggests that this cyclization takes place in a nonconcerted fashion via discrete carbon-centered radicals. Nevertheless, the termination step of the process seems to be subjected to a kind of water-dependent control, which is unusual in free-radical chemistry. The catalytic cycle is based on the use of the novel combination Me(3)SiCl/2,4,6-collidine to regenerate the titanocene catalyst. In practice this procedure has several advantages: it takes place at room temperature under mild conditions compatible with different functional groups, uses inexpensive reagents, and its end step can easily be controlled to give exocyclic double bonds by simply excluding water from the medium.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.