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Journal of Bacteriology, April 2001, p. 2646-2653, Vol. 183, No. 8
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.8.2646-2653.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Suppression of Hypersensitivity of Escherichia coli acrB Mutant to Organic Solvents by Integrational Activation of the acrEF Operon with the IS1 or IS2 Element

Kei Kobayashi,dagger Norihiko Tsukagoshi, and Rikizo Aono*

Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan

Received 21 August 2000/Accepted 17 January 2001

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrEF operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation. The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.

* Corresponding author. Mailing address: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan. Phone: 81 45-924-5966. Fax: 81 45-924-5819. E-mail: raono{at}bio.titech.ac.jp.

dagger Present address: Marine Biotechnology Institute, Shimizu-shi, Shizuoka 424-0037, Japan.

Journal of Bacteriology, April 2001, p. 2646-2653, Vol. 183, No. 8
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.8.2646-2653.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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