Some genetic and biochemical properties of the tetracycline resistance element of the Staphylococcus aureus plasmid pT181 have been studied. Resequencing of a portion of the tetracycline resistance gene (tet) showed the presence of a single open reading frame of 1,299 nucleotides capable of encoding a polypeptide of 433 amino acids. Analysis of BAL 31 nuclease-generated deletion mutants of the tet gene showed the presence of two complenientation groups within this region. Northern blot hybridizations demonstrated that the tet gene encodes a single mRNA, and its initiation site has been mapped by Sl nuclease protection experiments. We also identified an approximately 52,000-dalton tetracycline-inducible polypeptide in Bacilus subtilis miniicells carrying pT181. Induction of the tet gene by tetracycline resulted in a 4-fold increase in the levels of TET mRNA and at least a 15-fold increase in the amount of TET protein in B. subtilis minicells.Plasmid-mediated resistance to the tetracyclines (Tc) is a well-documented phenomenon within both gram-negative and gram-positive bacteria (9). The mechanism and genetic regulation of tetracycline resistance have been extensively studied in gram-negative species, specifically within the family Enterobacteriaceae (1,12,13,22,42). A number of studies have also been carried out on tetracycline resistance in gram-positive bacteria (4,9,10,15,16,25). Tetracycline resistance determinants identified within the streptococci and bacilli do not show significant homology with the determinants isolated from gram-negative bacteria at the DNA sequence level (5,15,26). The most common resistance mechanism appears to be energy-dependent efflux of the drug from resistant cells (9,24).In this study we describe characterization of the tetracycline resistance determinant of the Staphylococcus aureus plasmid pT181 (19). This plasmid is 4,437 base pairs (bp) in size and has been completely sequenced (20). Tetracycline resistance encoded by pT181 is induced by subinhibitory concentrations of tetracycline (19). Furthermore, the tet gene of pT181 shares considerable homology with the tet determinant of the Bacillus plasmids pTHT15 and pNS1981 at both the nucleotide and amino acid sequence levels (18,37 Bacterial strains were grown in CY broth and GL agar media as previously described by Novick and Brodsky (32). Growth media were supplemented with appropriate antibiotics at the following concentrations: tetracycline and erythromycin, 5 jig/ml; chloramphenicol, 3 jig/ml. Cells carrying pRN6010 were grown at 32°C (31). Induction with tetracycline was carried out by growing cells for 30 min, at which point a low dose of tetracycline (0.5 ,ug/ml) was added to the cultures. This was followed by another 30 min of growth and subsequent addition of a challenge dose of tetracycline (10 ,ug/ml).Enzymes. Pancreatic and T1 RNases were obtained from Sigma. Restriction enzymes, DNA polymerase I (Klenow fragment), bacterial alkaline phosphatase, S1 nuclease, and BAL 31 nuclease were purchased from Bethesda Research ...