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Journal of Bacteriology, November 1998, p. 5836-5843, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present in Mycobacterium fortuitum and Mycobacterium tuberculosis

José A. Aínsa,1,dagger Marian C. J. Blokpoel,2 Isabel Otal,1 Douglas B. Young,2 Koen A. L. De Smet,2 and Carlos Martín1,*

Departamento de Microbiología Medicina Preventiva y Salud Pública, Universidad de Zaragoza, 50009 Zaragoza, Spain,1 and Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St. Mary's Campus, London W2 1PG, United Kingdom2

Received 9 March 1998/Accepted 4 September 1998

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.

* Corresponding author. Mailing address: Departamento de Microbiología Medicina Preventiva y Salud Pública, Universidad de Zaragoza, C/Domingo Miral s/n, 50009 Zaragoza, Spain. Phone: 34-976-761759. Fax: 34-976-761664. E-mail: carlos{at}posta.unizar.es.

dagger Present address: Department of Genetics, John Innes Centre, Norwich NR4 7UH, United Kingdom.

Journal of Bacteriology, November 1998, p. 5836-5843, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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