Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis

Abstract

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance 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. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. 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 cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, 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 organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.

FIG. 1

Physical map of pCSA44 and its derivatives. pCSA44 contains the 15-kb DNA fragment with the tap_for gene (indicated by arrow) from M. fortuitum. Only the PstI sites delimiting the 2.3-kb fragment containing the tap_for gene are shown. The hatched area in plasmid pAC69 represents the deleted fragment.

FIG. 2

Map of the expression vector pSODIT-2. The thick black line denotes the region containing the mycobacterial origin of replication; the thin line denotes the E. coli origin of replication and ampicillin selection marker. Hyg, hygromycin selection marker; SOD, superoxide dismutase promoter.

FIG. 3

Alignment of the deduced M. fortuitum and M. tuberculosis Tap protein sequences. Identical amino acids are shown in black boxes. Various sequence motifs are shown in grey boxes under the alignment: motif GxLaDrxGrkxxl is characteristic of the MFS and is also described in the Prosite database as a sugar transport protein signature (accession no. PS00216); motif gxxxGPxxGGxl has been described as characteristic of drug export proteins; and motif GxxxGPL is characteristic of the 12-TMS family.

FIG. 4

Phylogenetic tree showing the relationships among different proteins homologous to the mycobacterial Tap proteins. A list of proteins analyzed is given in Table 3.

FIG. 5

Time course of tetracycline accumulation (ordinate) for M. smegmatis cells (open symbols) carrying the plasmid pSUM36 or pAC48 (tap_for gene). The closed symbols represent the same time course when the cells were preincubated for 60 min in the presence of CCCP.