CLONING OF AN ORGANIC SOLVENT-RESISTANCE GENE IN ESCHERICHIA-COLI - THE UNEXPECTED ROLE OF ALKYLHYDROPEROXIDE REDUCTASE

Although bacterial strains able to grow in the presence of organic sol vents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetr alin), a solvent with potential applications in industrial biocatalysi s, was used to select a resistant mutant of Escherichia coli. The resu ltant mutant strain was tested for resistance to a wide range of solve nts of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-di hydronaphthalene. A recombinant library from mutant DNA was used to cl one the resistance gene, The sequence of the cloned locus was determin ed and found to match the sequence of the previously described alkylhy droperoxide reductase operon ahpCF. The mutation was localized to a su bstitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzym e, The ahpC mutant was found to have an activity that was three times that of the wild type in reducing tetralin hydroperoxide to 1,2,3,4-te trahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the ce ll, The ahpC mutation increases the activity of the enzyme toward hydr ophobic hydroperoxides, thereby conferring resistance, The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, su ggesting that there are additional mechanisms of solvent toxicity, Mut ants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.