Cloning and characterization of a tetracycline resistance determinant present in Agrobacterium tumefaciens C58

Agrobacterium tumefaciens C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a m utation affecting a tRNA processing function significantly affected the eme rgence of such mutants, suggesting that C58 contained a positively acting g ene conferring resistance to tetracycline, A cosmid clone conferring resist ance to tetracycline in Escherichia coli and Agrobacterium was isolated fro m a genomic bank of one such mutant. Subcloning, transposon mutagenesis, an d DNA sequence analysis revealed that this DNA fragment contained two diver gently transcribed genes, tetA and tetR, encoding products that were very s imilar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant, tetR was disrupted by an IS426, The homologo us region from wild-type NT1 contained an intact tetR gene and did not conf er resistance to tetracycline, Hybridization analysis showed that of 22 mem bers of the genus Agrobacterium surveyed, only strains C58 and T37 containe d the tet determinant. Moreover, only these two strains mutated to resistan ce to this antibiotic. Unlike other Tet(A) systems, neither tetracycline no r a series of its derivatives induced the expression of this tet gene unit. Other polycyclic compounds, including many of plant origin, also did not i nduce this fet gene system. The divergent promoter region of this tet syste m contained a single inverted repeat element identical to one such operator repeat in the promoter region of the tet determinant from the IncP1 alpha R plasmid RP4. TetR repressor proteins from the Agrobacterium tet system an d from RP4 interacted with the heterologous operators, While the repressive effect of the TetR protein from strain C58 (TetR(C58)) on the tetA gene fr om strain RP4 (tetA(RP4)) was not relieved by tetracycline, repression of t etA(C58) by Tet(RP4) was lifted by this antibiotic.