We used the TnlO-encoded Tet repressor, which has a highly specific binding capacity for tetracycline, to probe contacts between the drug and protein by chemical interference studies of the antibiotic. For that purpose, the equilibrium association constants Resistance to tetracycline (Tc) in enterobacteria is mostly mediated by active efflux of the drug from the resistant cell. This property can be encoded by five related genetic determinants, named classes A through E, which share extensive sequence homology (9). The most frequently found determinant is located on transposon TnJO. Resistance to Tc mediated by TnJO is inducible on the level of transcription (2). Repression is mediated by a repressor protein (1, 13) which binds as a dimer (5) to two operator sequences in the tet regulatory region (7). The inducer is the antibiotic Tc itself, which interacts with the repressor-operator complex, leading to at least a 1,000-fold reduction in operator binding affinity (5,8). Since Tc inhibits protein synthesis in the bacterial cell (4), the release of repression must be very efficient to allow the synthesis of the resistance protein.Tc-Tet repressor binding has been studied by nitrocellulose filter retention (6) and fluorescence measurements (16

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A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 bp synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor.

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