ANALYSIS OF THE AAC(3)-VIA GENE ENCODING A NOVEL 3-N-ACETYLTRANSFERASE

Biochemical analysis (G . A. Papanicolaou, R. S. Hare, R. Mierzwa, and G. H. Miller, abstr. 152, Program Abstr. 29th Intersci. Conf. Antimic rob. Agents Chemother., 1989) demonstrated the presence of a novel 3-N -acetyltransferase in Enterobacter cloacae 88020217. This organism was resistant to gentamicin, and the MIC of 2'-N-ethylnetilmicin for it w as fourfold lower than that of 6'-N-ethylnetilmicin, a resistance patt ern which suggested 2'-acetylating activity. However, high-pressure li quid chromatography analysis demonstrated that the enzyme acetylated s isomicin in the 3 position. We have cloned the structural gene for thi s enzyme from a large (>70-kb) conjugative plasmid present in E. cloac ae. Subcloning experiments have localized the aac(3)-VIa gene to a 2.1 -kb Sau3A fragment. The deduced AAC(3)-VIa protein showed 48% amino ac id identity to the AAC(3)-IIa protein and 39% identity to the AAC(3)-V II protein. Examination of the 5'-flanking sequences demonstrated that the aac(3)-VIa gene was located 167 bp downstream of the aadA1 gene a nd was present in an integron. In addition, the aac(3)-VIa gene is als o downstream of a 59-base element often seen in an integron environmen t. Primer extension analysis has identified a promoter for the aac(3)- VIa gene downstream of both the aadA1 gene and a 59-base element.