Biochemical and genetic characterization of the tet determinant of Bacillus plasmid pAB124

Abstract

A fragment of Bacillus plasmid pAB124 carrying the genes encoding tetracycline resistance was previously cloned into Escherichia coli plasmid pSF2124 (S.J. Eccles, A. Docherty, I. Chopra, S. Shales, and P. Ball, J. Bacteriol. 145:1417-1420, 1981). The cloned pAB124 tet fragment conferred low-level resistance in E. coli, but exposure of this strain to a subinhibitory level of tetracycline led to selection of a mutant plasmid in which high-level resistance, associated with decreased drug accumulation, was expressed constitutively. In this plasmid, the Bacillus tet determinant appeared to be transcribed from a promoter on the vector. Construction of tetracycline-sensitive derivatives of this plasmid by transposon insertion mutagenesis allowed identification of a 32,000-dalton membrane-located protein, which apparently promoted decreased accumulation of tetracycline. This protein was also synthesized as a 32,000-dalton polypeptide in a coupled, in vitro transcription-translation system directed by plasmid DNA. The pAB124 tet determinant differed from the tetA through tetD determinants found in gram-negative bacteria in DNA-DNA hybridization and in the ability to prevent accumulation of different tetracycline derivatives, but was closely related to the tet determinant of another plasmid isolated from Bacillus species, pBC16.