Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present inMycobacterium fortuitumandMycobacterium tuberculosis

Publisher Website
Google Scholar
Abstract
A recombinant plasmid isolated from aMycobacterium fortuitumgenomic library by selection for gentamicin and 2-N′-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced intoM. smegmatis. Further characterization of this plasmid allowed the identification of theM. fortuitum tapgene. A homologous gene in theM. tuberculosisH37Rv genome has been identified. TheM. tuberculosis tapgene (Rv1258 in the annotated sequence of theM. tuberculosisgenome) was cloned and conferred low-level resistance to tetracycline when introduced intoM. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function 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 protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing thetapgenes were decreased. When tetracycline accumulation experiments were carried out with theM. fortuitum tapgene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organismM. fortuitumand the important pathogenM. tuberculosisare probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.