Development of two DNA probes for differentiating the structural genes of subclasses I and II of the aminoglycoside-modifying enzyme 3'-aminoglycoside phosphotransferase

Abstract

Two DNA probes were developed to screen for the genes encoding 3'-aminoglycoside phosphotransferase activity in gram-negative bacilli. The 3'-I phosphotransferase [APH(3')I] probe was subcloned from Tn903; the APH(3')II probe was subcloned from Tn5. Each probe proved to be specific for genes corresponding to its own APH(3') subclass and did not hybridize with DNA from other classes when tested at high stringency by either Southern hybridization or dot-blot hybridization methods. The APH(3')I probe hybridized to DNA obtained from organisms demonstrating APH(3')I activity as measured by the phosphocellulose paper binding assay (PPBA) as well as to DNA from organisms reported to have both APH(3')I and APH(3')II activity by PPBA. This probe did not hybridize to DNA from organisms showing only APH(3')II activity by PPBA. The APH(3')II probe demonstrated homology with DNA from organisms showing APH(3')II activity by PPBA but not with DNA from organisms showing APH(3')I activity or both APH(3')I and APH(3')II activity by PPBA. We conclude that organisms previously believed to contain both APH(3')I and APH(3')II genes based on PPBA contain in fact only the APH(3')I gene.