Identification of repressor binding sites controlling expression of tetracycline resistance encoded by Tn10

Abstract

The regulatory region controlling the expression of tetracycline resistance and repressor genes contains two nearly identical regions of dyad symmetry. Deletions of this control region were isolated by digestion with S1 nuclease. The ability of these deletions to bind the tet repressor was determined by an in vivo repressor titration assay. The results indicate that repressor specifically binds both regions of dyad symmetry.

Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant

“…However, isogenic tetR+ tetA-lacZ and tetR+ tetR-lacZ strains are equally responsive to tetracycline. This result is consistent with a model in which tetA and tetR transcription are coordinately regulated by repressor binding to shared operator sites that overlap the divergent tetA and tetR promoters (7,29,30,57).…”

“…These results are qualitatively similar to the results obtained previously with tet-lac translational fusion strains (5,6). In addition, the high-level synthesis of ,-galactosidase in lysogens of the tetA-lacZ phage ARStetl58-43, and its repression by tetR+ plasmids, confirms that the tetA promoter-operator region is within the 158-bp TaqI fragment that spans the region between the tetA and tetR structural genes (7,28,34,56,57). Lastly, the capacity of plasmid subclones carrying the 701-bp HinclI fragment to regulate P-galactosidase synthesis in tet-lac strains confirms that the tetR structural gene is within the 701-bp HincII fragment (6,16,28,56) and in addition provides a sensitive assay for the analysis of tetR and tet operator mutations (47,57).…”

“…The tetA gene encodes a 43.2-kilodalton membrane protein that appears to be both necessary and sufficient for resistance to tetracycline (29,34,37,49). The tetR gene encodes a 23.3-kilodalton protein that negatively regulates both its own synthesis and the synthesis of the TetA resistance protein (6,28,57,58); tetracycline induces synthesis of both the TetA resistance protein and the TetR repressor (6,34,37,56). The TetR repressor has been purified and shown to bind to appropriate tet DNA fragments in the absence, but not in the presence, of tetracycline (28).…”

Use of transcriptional repressors to stabilize plasmid copy number of transcriptional fusion vectors

“…The lacUV5 promoter, cloned into pRZ5245, is a catabolite activating protein-independent mutant which has strong transcriptional activity. The strong tetA promoter was cloned into pBR322 by Wray and Reznikoff (16). In this construct, pRT301, the tetA promoter does not direct the expression of an assayable gene such as lacZ; however, the effect of the strong promoter on plasmid stability can still be observed and studied.…”

“…2C). However, when the compatible plasmid pRT241 (16) which contains the tetracycline repressor (tetR) gene is introduced into the above strain, pRT301 is stably maintained (Fig. 2C).…”