Differential regulation of the Tn10-encoded tetracycline resistance genes tetA and tetR by the tandem tet operators O1 and O2.

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RESUMO

The Tn10-encoded tet transcriptional control sequence consists of bidirectional, overlapping promoters which are superimposed by a tandem operator arrangement. Three mutations have been constructed by oligonucleotide-directed mutagenesis which reduce binding of Tet repressor to either one or both of the tandem tet operators 1000-fold as determined by DNAseI footprinting in vitro. The affinity of Tet repressor for mutant tet operators is not affected by the presence of an already occupied neighbouring wild-type operator, indicating little or no cooperativity. The regulation of the divergently oriented tet promoters PA and PR by the tet operators O1 and O2 and Tet repressor provided in trans is determined using transcriptional fusions of the promoters to lacZ and galK indicator genes located with different polarity on the same plasmid. The results demonstrate that expression of the resistance gene tetA is regulated by Tet repressor bound to either O1 or O2. Expression of the repressor gene tetR is only marginally reduced when Tet repressor is bound to O2. This result is discussed with respect to the double promoter structure found for PR. Occupation of O1 with Tet repressor turns off transcription from PR completely. The implications of these findings on the establishment of tetracycline resistance upon induction are discussed.

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