title: Tet protein domains interact productively to mediate tetracycline resistance when present on separate polypeptides.
abstract: Both domains, alpha and beta, of the cytoplasmic membrane-localized Tet proteins encoded by the tet gene family (classes A through E) are required for resistance to tetracycline (Tcr) in gram-negative bacteria. Two inactive proteins, each containing a mutation in the opposite domain, are capable of complementation to produce Tcr. Similarly, inactive hybrid proteins expressed by interdomain gene hybrids constructed between tet(B) and tet(C) [tet(B) alpha/(C) beta and tet(C) alpha/(B) beta] together produce significant Tcr via trans complementation (R.A. Rubin and S. B. Levy, J. Bacteriol. 172:2303-2312, 1990). A derivative of tet(B) was constructed to express the two domains of Tet(B) as separate polypeptides, neither containing intact the central, hydrophilic interdomain region. Cells harboring this tet(B) mutant expressed Tcr at about 20% the level conferred by intact tet(B). As expected, no detectable amount of a full-length Tet protein was expressed. A polypeptide corresponding to the alpha domain was observed. Interdomain hybrids between tet(B) and tet(C) containing a frameshift at the fusion junction, designed to result in expression of each of the four domains on separate polypeptides, showed trans complementation without production of detectable full-length proteins. Levels of Tcr were greater than or equal to those previously observed in complementations using full-length hybrid proteins. These results strongly suggest that polypeptides harboring individual alpha and beta domains, lacking an intact interdomain region, can interact productively in the cell to confer Tcr.
authors.affiliation: Department of Molecular Biology, Tufts University School of Medicine, Boston, Massachusetts.
authors.forename: R A
authors.initials: RA
authors.lastname: Rubin
authors.forename: S B
authors.initials: SB
authors.lastname: Levy
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D016368:Frameshift Mutation; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005838:Genotype; D046911:Macromolecular Substances; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011815:R Factors; D012097:Repressor Proteins / Q000235:genetics*; D015183:Restriction Mapping; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics*
chemical_list: D046911:Macromolecular Substances; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline