MOLECULAR-CLONING AND CHARACTERIZATION OF TAP, A PUTATIVE MULTIDRUG EFFLUX PUMP PRESENT IN MYCOBACTERIUM-FORTUITUM AND MYCOBACTERIUM-TUBERCULOSIS

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistanc e conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tube rculosis genome) was cloned and conferred low-level resistance to tetr acycline when introduced into Ill. smegmatis. The sequences of the put ative Tap proteins showed 20 to 30% amino acid identity to membrane ef flux pumps of the major facilitator superfamily (MFS), mainly tetracyc line and macrolide efflux pumps, and to other proteins of unknown func tion but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonoph ore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of res istance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carrie d out with the M. fortuitum tap gene, the level of tetracycline accumu lation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunis tic organism M. fortuitum and the important pathogen M. tuberculosis a re probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.