title: In vivo activation of tetracycline repressor by Cre/lox-mediated gene assembly.
abstract: Tetracycline repressor (TetR) bears an unstructured loop region between helices alpha8 and alpha9, 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 TetR(lox72/1), is distinguished from wt-TetR by a slightly elongated and altered alpha8-alpha9 loop only, harboring an amino acid stretch encoded by lox72. Despite decreased intracellular protein amounts, TetR(lox72/1) 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 alpha8-alpha9 loop and demonstrate the possible use of the regulator as a read-out tool for the activity of site-specific recombinases.
authors.affiliation: Lehrbereich mikrobielle Genetik, Eberhard-Karls-Universität Tübingen, Tübingen, Deutschland. ralph.bertram@uni-tuebingen.de
authors.forename: Ralph
authors.initials: R
authors.lastname: Bertram
authors.forename: Martina
authors.initials: M
authors.lastname: Kolb
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D019426:Integrases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000096:biosynthesis*; D013753:Tetracycline Resistance
chemical_list: D012097:Repressor Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases