Nucleic Acids Research

The nucleotide sequence of a transposon Tn7 DNA fragment encoding a 3"(9)-O-nucleotidyltransferase, an aminogiycoside-modifying enzyme, which mediates bacterial resistance to spectinomycin and streptomycin, was determined. The aadA structural gene was 786 bases long and predicted a polypeptide of 262 amino acids with a calculated molecular weight of 29,207. Comparison of the DNA sequences of Tn7 and plasmid R538-1 indicated that their aadA genes were nearly identical. Comparison of the polypeptides predicted by the aadA genes of Tn7 and Tn554 indicated that the genes were related. INTRODUCTION Bacterial resistance to spectinomycin (Sp) and streptomycin (Sm) can be mediated by plasmid-specified genes that code for aminoglycosidemodifying enzymes (1). Streptomycin adenylyltransferase is a 3"(9)-Onucleotidyltransferase, AAD(3")(9), which adenylylates either the 3"-hydroxyl on the amino-hexose III ring of streptomycin or the 9-hydroxyl on the actinamine ring of spectinomycin (2). The AAD(3")(9) enzyme has been found in both Gram-negative and Gram-positive genera (3). Spectinomycin adenylyltransferase, a 9-0-nucleotidyltransferase, AAD(9), adenylylates only the 9-hydroxyl on the actinamine ring of spectinomycin (2). The AAD(9) enzyme is the product of a gene that is present on transposon Tn554, which is found in Staphylococcus aureus (14). The dual substrate profile of the AAD (3")(9) enzyme is common, suggesting that the aadA gene is widespread. One source of the aadA gene, which will be considered in this study, is plasmid R538-1, a relative of plasmid R100 (5). On plasmid R538-1 or R1O0, the aadA gene is linked to a sulfonamide-resistance determinant. It is disseminated as part of Tn2l or any of several related complex transposons that are present in plasmids R100, Rl, R5, R6, or NRl (6,7,8). Another distinct source of the aadA gene is the transposon Tn7, originally isolated from the R-plasmid © I R L Press Limited, Oxford, England. 7095 Nucleic Acids Research R483 (9). On Tn7 the aadA gene is linked to the Type I dihydrofolate reductase gene, which confers resistance to the antibiotic trimethoprim. To study the aadA gene of transposon Tn7, the nucleotide seauence was determined. The nucleotide sequences and deduced polypeptides of the Tn7. Tn554, and R538-1 aadA genes were compared. The evolution of the aadA genes of both Gram-negative and Gram-positive origin is discussed. MATERIALS AND METHODS Bacterial Strains and Plasmids The bacterial strain E coliK12, C600. concaining plasmid pFE506 (10) was used to make all deletion constructions and to clone the aadA gene. The cloning vectors, pUC8 and pUC9, their host JM83, the bacteriophages M13mp8 and M13mp9, and their host JM103 were acquired through Bethesda Research Laboratories. Inc. Gaithersburg, MD, and propagated as previously described (21,12). Antibiotic Sensitivity Determination Minimal inhibitory concentrations (MIC) for Sp were determined by a standard agar dilution method (13). Molecular Cloning, Clone Analysis, and Gel Electrophoresis Hybrid plasmids and bacteriophages were constructed using techniques previously described (14). DNA fragments were isolated from agarose gels by electrophoresis onto NA45 paper (15). Nucleotide Sequence Determination The chain termination technique of DNA sequencing (16) was used. Templates were generated as previously described (14). Sequencing reagents supplied as a kit from New England Nuclear Corp., Boston, MA, were supplemented by a 17-mer primer (Pharmacia P.L. Biochemical, Milwaukee, WI) and the Klenow fragment of polymerase I (Bethesda Research Laboratories, Inc.). Buffer gradient sequencing gels were prepared as described by Biggin et.al. (17). Compressions of the sequencing products during gel electrophoresis were resolved by substituting dITP for dGTP (18). The concentration of dITP was 50 pM in the I and A reactions, and 65 pM in the C and T reactions.