Ferrante A A, Augliera J, Lewis K, Klibanov A M
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7617-21. doi: 10.1073/pnas.92.17.7617.
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.
尽管已经分离出了能够在有机溶剂存在下生长的细菌菌株，但对其抗性机制却知之甚少。在本研究中，1,2,3,4-四氢萘（萘满），一种在工业生物催化中有潜在应用的溶剂，被用于筛选大肠杆菌的抗性突变体。对所得突变菌株进行了对多种不同疏水性溶剂的抗性测试，发现它不仅对萘满本身有抗性，而且对环己烷、丙苯和1,2-二氢萘也有抗性。利用突变体DNA构建的重组文库来克隆抗性基因。测定了克隆位点的序列，发现其与先前描述的烷基过氧化氢还原酶操纵子ahpCF的序列匹配。该突变定位在ahpC编码区第142位的甘氨酸被缬氨酸取代，ahpC是编码该酶催化亚基的基因。发现ahpC突变体在将萘满氢过氧化物还原为1,2,3,4-四氢-1-萘酚方面的活性是野生型的三倍。我们得出结论，萘满等此类溶剂的毒性是由细胞内有毒氢过氧化物的形成引起的。ahpC突变增加了该酶对疏水性氢过氧化物的活性，从而赋予抗性。ahpC突变体对亲水性更强的溶剂二甲苯和甲苯敏感，这表明存在其他溶剂毒性机制。从ahpC突变体中分离出了对二甲苯和萘满混合物有抗性的突变体，但从野生型菌株中未分离出。
