The expression of plasmid-encoded tetracycline resistance (tet) genes in Escherichia coli K12 and other enteric bacteria is induced by tetracycline. Recent work has focused on the distantly related tet genes encoded by the transposon Tn10 and the plasmid pSC101. Tn10 and pSC101 tet both consist of a resistance gene (tetA) and a repressor gene (tetR) that are transcribed from divergent overlapping promoters. The tetR gene product represses transcription of tetA and tetR, and tetracycline coordinately induces transcription of tetA and tetR.
The specific aims of this proposal are to characterize, by genetic and biochemical analysis, the transcriptional control elements that regulate expression of the Tn10 and pSC101 tet genes. Comparative analysis of these distantly related systems--less than 50% DNA sequence homology--will provide insights into structure-function relationships that would be difficult to obtain by studying either system alone. In particular: (1) Recent work suggests that the Tn10 tetA promoter activates one or both of the overlapping tetR promoters. The molecular details of this novel mechanism of promoter activation will be examined. (2) The functional sites of tet repressor will be localized by genetic analysis, including the isolation of Tn10 tetR point mutations that affect operator or inducer binding, and the construction of hybrid Tn10-pSC101 tet repressors. (3) The pSC101 tet repressor will be purified by procedures similar to those used for the purification of Tn10 repressor. (4) The interaction of purified Tn10 and pSC101 tet repressors with tetracycline and operator DNA will be studied by fluorescence spectroscopy. (5) Finally, the DNA sequence of the class E tet genes carried on the Salmonella ordonez plasmid pIP173 will be determined, in order to extend existing sequence data on distantly related tet genes. These studies will make extensive use of tet-lac gene fusions, oligonucleotide-directed in vitro mutagenesis, DNA and RNA sequencing, and in vitro transcription methodologies. The long-term objective of these studies is to increase understanding of the molecular components and mechanisms involved in the regulation of prokaryotic genes in general, and plasmid-encoded tetracycline resistance genes in particular.
