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Antimicrobial Agents and Chemotherapy, August 2003, p. 2413-2417, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2413-2417.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Cloning and Characterization of an ABC Multidrug Efflux Pump, VcaM, in Non-O1 Vibrio cholerae

Nazmul Huda, Eun-Woo Lee, Jing Chen, Yuji Morita, Teruo Kuroda, Tohru Mizushima, and Tomofusa Tsuchiya*

Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan

Received 24 February 2003/ Returned for modification 17 April 2003/ Accepted 13 May 2003

A gene responsible for multidrug resistance was cloned from the chromosomal DNA of non-O1 Vibrio cholerae NCTC 4716 by using as a host drug-hypersensitive Escherichia coli strain KAM32, which lacks major multidrug efflux pumps. E. coli cells transformed with the gene showed elevated levels of resistance to a number of structurally dissimilar drugs, such as tetracycline, norfloxacin, ciprofloxacin, doxorubicin, daunomycin, 4',6-diamidino-2-phenylindole, and Hoechst 33342. We determined the nucleotide sequence and found one open reading frame. We designated the gene vcaM. The deduced product, VcaM, seems to be a polypeptide with 619 amino acid residues (69 kDa) that has a putative topology of six transmembrane segments in the N-terminal hydrophobic domain, followed by an ATP binding domain in the C-terminal hydrophilic region. The sequence of VcaM was shown to be similar to those of human multidrug resistance proteins P-glycoprotein MDR1 and lactococcal LmrA, which are driven by ATP. The efflux of Hoechst 33342 and doxorubicin from cells possessing VcaM was detected. The efflux activity was inhibited by reserpine and sodium o-vanadate, which are potent inhibitors of MDR1 and LmrA. Thus, we conclude that VcaM is a member of the family of multidrug efflux pumps of the ATP binding cassette type and the first experimentally proven example of a multidrug efflux pump of this family in gram-negative bacteria.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan. Phone and Fax: 81-86-251-7957. E-mail: tsuchiya{at}pharm.okayama-u.ac.jp.

Antimicrobial Agents and Chemotherapy, August 2003, p. 2413-2417, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2413-2417.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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