Identification of the tetracycline resistance promoter and repressor in transposon Tn10

Abstract

The structural and regulatory functions encoding tetracycline resistance in transposon Tn10 lie within a 2,700-base pair region. Using recombinant plasmids with different deoxyribonucleic acid sequences adjacent to a HincII site in this region, we located the promoter controlling the expression of tetracycline resistance. These various sequences conferred altered levels of tetracycline resistance. Plasmids containing deletions of a 695-base pair HincII fragment were constitutive and showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thus identifying the repressor and the location of its gene.

A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance

“…Regulatory mutants of TnlO that express tetracycline resistance constitutively have been isolated (10)(11)(12). Most of the data available are consistent with a model of negative control (6,40,41), although the observation that regulatory mutants of TnlO often have a dominant constitutive phenotype may indicate that a more complex type of control exists (11). To investigate the regulation of the tetracycline resistance of TnlO in detail, we isolated mutants defective in the regulation of resistance.…”

“…Evidence for the existence of a regulatory gene for tetracycline resistance was provided by mutant analyses, gene fusions, and regulatory studies. From the phenotypes of deletion mu-tants generated by restriction enzymes, it was concluded that the regulatory region is located on the left side of the tetA gene (10,40), as shown in Fig. 1.…”

Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification.

“…This is particularly true for regulatory proteins which negatively control their own synthesis like the cIgene product from the Escherichia coli phage X (Johnson et al, 1980). Negative control of its own expression has also been reported for the Tn 10-encoded TET repressor which regulates the expression of the tetracycline resistance operon on TnIO (Beck et al, 1982;Wray et al, 1981;Hillen et al, 1982). The control of transcription in this operon is of particular interest because it is complex with overlapping divergent promoters (Bertrand et al, 1983;Hillen et al, 1984) and a tandem arrangement of nearly identical operator sequences separated by 12 bp .…”

“…Due to its negative control, early attempts to prepare the repressor protein were only partially successful (Yang et 1976). Even the construction of a constitutive overproducer of the TET repressor employing a weak promoter from Tn5 (Wray et al, 1981) did not result in purification of the TET repressor to homogeneity (Hillen et al, 1982).…”

“…Figure 1 shows part of the TnlO DNA containing the TET repressor gene. It is located between HincII sites on a 695-bp DNA fragment (Wray et al. 1981).…”