Expression of the tetK gene from Staphylococcus aureus in Escherichia coli: comparison of substrate specificities of TetA(B), TetA(C), and TetK efflux proteins

Abstract

The tetK gene, which encodes a tetracycline efflux pump from Staphylococcus aureus, was expressed in Escherichia coli by using an inducible, low-level expression system. The tetK gene, as well as the tetA(B) gene from the transposon Tn10 and the tetA(C) gene from plasmid pBR322, was subjected to the regulatory control of the lac repressor, and resistance to tetracycline was measured as a function of the isopropyl-beta-D-thiogalactopyranoside concentration. The maximum resistance of the E. coli strain containing the tetK construct was comparable to the maximum resistance of the strain containing the tetA(C) construct but was less than the resistance of the strain containing the tetA(B) construct. Overexpression of the tetK, tetA(B), or tetA(C) genes was toxic. When expression was regulated so that resistance to tetracycline was comparable, then the TetA(B) and TetA(C) proteins conferred very similar levels of resistance to a variety of tetracycline derivatives. In contrast, the TetK protein was less capable of conferring resistance to the tetracycline derivatives minocycline, 6-deoxy-6-demethyltetracycline, and doxycycline. The implications for the recognition of various tetracycline substituents by the TetK protein are discussed.