Nucleotide Sequence of the Mm Tetracydine Resistance Determinant of the Streptococcal Conjugative Shuttle Transposon Tnls4s

The nucleotide sequence of the tetracycline resistance gene tetM encoded by streptococcal conjugative shuttle transposon Tnl545 has been determined. The resistance gene was identified as a coding sequence of 1917 base pairs corresponding to a protein with a Mj. of 72,500 daltons. This value is in good agreement with that, 68,000 daltons, estimated by SDS-polyacrylamide gel electrophoresls of Escherichia coll minicell extracts. The tetM gene product does not exhibit any sequence homology with either the Gram-negative UetA, tetB and tetC), or the Bacillus and Staphylococcus tetracycline resistance proteins. The average hydropathy value of the tetM gene product (-0.21) contrasts with those calculated for the other TET proteins which are markedly hydrophoblc (0.76 to 0.93). Hybridization experiments performed with an intragenlc tetM probe do not support the claim (Taylor, D. (1986), J. Bact. 165, 10371039)) that tetracycline resistance in Campylobacter Is due to acquisition of tetM. INTROOUCTK3N Tetracycline (Tc) belongs to a family of broad spectrum antibiotics which disrupt protein synthesis by interfering with the binding of the ternary aminoacyltRNA.EFTu.GTP complex to the acceptor site of the ribosome (1). Tetracycline resistance (Tcfy is the most common antibiotic resistance encountered in nature. In Gram-negative bacteria, a minimum of four genetically different classes of Tc^ genes (tetA, tetB, tetC and tetD) have been defined (2). These genes are generally borne by plasmids or transposons and code for a Tc-inducible hydrophobic membrane-located polypeptide which promotes an energy-dependent efflux of the antibiotic from the cell (1,3). In the Gram-positive streptococci, three Tc^ determinants (tetL, tetM and tetN) have been identified (4) which are not structurally related to the genes from Gramnegative bacteria (2,4). The tetL determinant is widespread among such evolutlonarlly distant Gram-positive bacteria as Streptococcus, Bacillus and Staphylococcus (5,6) and probably mediates the active efflux of Tc (7). By contrast, in streptococcal strains containing either the tetN or tetM determinants, TcR is mediated at the level of protein synthesis (7). A similar mechanism of resistance to Tc has been also reported in Streptomyces aureofaciens (8) and in S. rlmosus (9) which produce oxytetracycline. © IR L Press Limited, Oxford, England. 7 0 4 7 Nucleic Acids Research While tetN seems to be rare, tetM is apparently dispersed in various bacterial genera, including pathogens responsible for sexually transmitted diseases, such aa Mycoplaama homlnia, Ureaplasma urealyticum and Gardnerella vaginalis (3,10-16). The fact that tetM determinant is frequently borne by conjugative transposable elements may account for this broad distribution. The conjugative shuttle transposon Tnl545 has been detected in the chromosome of a pneumococcal strain (17). This 25.3-kb element confers resistance to kanamycin by synthesis of a 3'-aminoglycoside phosphotransferase of type III (aphA-3), to macrolldeHnco8amide-8treptogramln B-type antibiotics (ermAM) and to tetracycllne (tetM) (Courvalin and Carlier, submitted). We report here the nucleotide sequence of the resistance gene tetM and the characterization of the corresponding gene product, the TETM protein, in minicella of E_. coli. A tetM-specific probe consisting of an intragenlc tetM fragment cloned into E. coli phage M13 was constructed and used in dot blot hybridization experiments to study the distribution of this gene in phylogenetically remote pathogenic bacteria. MATERIALS AND METHODS a) Bacterial strains, plasmida and phagns E_. coli strain JM101 was transfected with DNA fragments to be sequenced, which had been cloned in bacteriophage M13mpl8, M13mpl9 (18) or M13mplO (19) purchased from Pharmacia-PL Biochemlcals Inc. Recombinant plasmlds were introduced by transformation (20) into E. coÛ HB101 (21) and E. ccM AR1062 (22), a minicellproducing strain. Antibiotic concentrations for selection were amplcillin, lOOpg/ml; kanamycin, 20ug/ml; and tetracycline, 5pg/ml. Transposon Tnl545 DNA was prepared from S. faecalis strain BM4127 harboring pIP804 (pIP964::Tn!545) (Courvalin and Carlier, submitted). Plasmid pAT181 (Ap, Km , TcS) constructed by the in vitro deletion of the BamHI-Aval fragments of pAT95 (pBR322nKm) (23), was used for cloning experiments. b) Preparation of plasmid DNA Isolation of pIP804 DNA (24) and the large-scale (20) and small-scale (25) preparations of plasmid DNA from E. coli were as described. c) Analysis and purification of DNA fraqjiwnta Restriction fragments were analyzed by 0.8% slab agarose gel (10xl0x0.7cm) electrophoresis using Trls-Borate-EDTA buffer. DNA restriction fragments were recovered from preparative gels by electrotransfer onto dialysis membranes. d) Mucleotide sequencing DNA fragments cloned into bacteriophage M13 derivatives were sequenced by the dideoxynucleotide chain terminator technique (26). DNA fragments were resolved by