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Applied and Environmental Microbiology, August 2004, p. 4424-4431, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4424-4431.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp.

Hiroko Tokunaga,¹ Kenjiro Mitsuo,¹ Sachiyo Ichinose,² Akira Omori,² Antonio Ventosa,³ Taiji Nakae,⁴ and Masao Tokunaga^1*

Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065,¹ Laboratory of Protein, Structure Analysis Section, Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194-8511,² Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan,⁴ Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, E-41012 Seville, Spain³

Received 2 October 2003/ Accepted 11 March 2004

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.

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* Corresponding author. Mailing address: Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. Phone and fax: 81-99-285-8634. E-mail: tokunaga{at}chem.agri.kagoshima-u.ac.jp.

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Applied and Environmental Microbiology, August 2004, p. 4424-4431, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4424-4431.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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