In vivo Activation of Tetracycline Rep ressor by Cre/lox-Mediated Gene Assembly

Tetracycline repressor (TetR) bears an unstructured loop region between helices α8 and α9, which is moderately permissive to amino acid exchanges and length variations. Recognition sites for the site-specific recombinases Flp (FRT) or Cre (lox) were inserted in-frame into tetR, substituting some of this loop’s codons. A number of the deduced TetR variants displayed efficient regulation in vivo, thus allowing the establishment of a new mode of TetR activation on the genetic level. Chromosomally encoded tetR in Bacillus subtilis was disrupted and inactivated by insertion of a lox66-aphAIII-lox71 kanamycin resistance cassette. Marker excision by Cre recombinase led to the assembly of a novel tetR allele. The encoded regulator, termed TetRlox72/1, is distinguished from wt-TetR by a slightly elongated and altered α8–α9 loop only, harboring an amino acid stretch encoded by lox72. Despite decreased intracellular protein amounts, TetRlox72/1 displayed efficient in vivo activity in B. subtilis and E. coli, indistinguishable from that of wt-TetR. These results underline the sequence flexibility of TetR in the α8-α9 loop and demonstrate the possible use of the regulator as a read-out tool for the activity of site-specific recombinases.

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