Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.
Although bacterial strain able to grow in the presence of organic solvents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetralin), a solvent with potential applications in industrial biocatalysis, was used to select a resistant mutant of Escherichia coli. The resultant mutant strain was tested for resistance to a wide range of solvents of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene. A recombinant library from mutant DNA was used to clone the resistance gene. The sequence of the cloned locus was determined and found to match the sequence of the previously described alkylhydroperoxide reductase operon ahpCF. The mutation was localized to a substitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzyme. 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-tetrahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the cell. The ahpC mutation increases the activity of the enzyme toward hydrophobic hydroperoxides, thereby conferring resistance. The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, suggesting that there are additional mechanisms of solvent toxicity. Mutants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.
Ferrante
A A
AA
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Augliera
J
J
Lewis
K
K
Klibanov
A M
AM
D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009876:Operon; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D010544:Peroxidases; D054464:Peroxiredoxins; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017384:Sequence Deletion; D012997:Solvents / Q000494:pharmacology*; D013764:Tetrahydronaphthalenes / Q000494:pharmacology*
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012997:Solvents; D013764:Tetrahydronaphthalenes; D010088:Oxidoreductases; D010544:Peroxidases; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; C095210:tetralin