The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis. However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2′,7′-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters.
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October 2002
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Research Article|
October 01 2002
Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis Available to Purchase
Baisakhee Saha CHOUDHURI;
Baisakhee Saha CHOUDHURI
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India
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Sanjib BHAKTA;
Sanjib BHAKTA
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India
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Rajib BARIK;
Rajib BARIK
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India
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Joyoti BASU;
Joyoti BASU
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India
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Manikuntala KUNDU;
Manikuntala KUNDU
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India
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Parul CHAKRABARTI
Parul CHAKRABARTI
1
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
April 18 2002
Revision Received:
May 21 2002
Accepted:
June 11 2002
Accepted Manuscript online:
June 11 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 367 (1): 279–285.
Article history
Received:
April 18 2002
Revision Received:
May 21 2002
Accepted:
June 11 2002
Accepted Manuscript online:
June 11 2002
Citation
Baisakhee Saha CHOUDHURI, Sanjib BHAKTA, Rajib BARIK, Joyoti BASU, Manikuntala KUNDU, Parul CHAKRABARTI; Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis. Biochem J 1 October 2002; 367 (1): 279–285. doi: https://doi.org/10.1042/bj20020615
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