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Identification, cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from Vibrio cholerae O395

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Abstract

A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 μg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 μg/ml), 62.5-fold for rifampin (MIC = 50 μg/ml), >30-fold for erythromycin (MIC = 50 μg/ml) and minocycline (MIC = 2 μg/ml), 20-fold for trimethoprim (MIC = 0.12 μg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 μg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 μg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 μg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.

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Acknowledgments

This work was made possible by NIH Grants 1 R15 GM070562-04 and 2 P20 RR016480-09, the latter of which is from the NM-INBRE program of the National Center for Research Resources, a contribution from Calton Research Associates in honor of George and Clytie Calton, and an Internal Research Grant awarded by Eastern New Mexico University. We thank Dr. Jeffrey K. Griffith (University of New Mexico, Albuquerque, NM) and Dr. Thomas H. Wilson (Harvard Medical School, Boston, MA) for helpful comments. We thank Dr. Tomofusa Tsuchiya (Laboratory of Molecular Microbiology, University of Okayama, Japan) for E. coli strain KAM32, and Dr. Chythanya Rajanna (Emerging Pathogens Institute, Gainesville, FL) for Vibrio cholerae O395.

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  1. Department of Biology, Eastern New Mexico University, Roosevelt Hall, Room 101, Station 33, Portales, NM, 88130, USA

    Kenneth P. Smith, Sanath Kumar & Manuel F. Varela

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  1. Kenneth P. Smith
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Correspondence to Manuel F. Varela.

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Communicated by Jorge Membrillo-Hernández.

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Smith, K.P., Kumar, S. & Varela, M.F. Identification, cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from Vibrio cholerae O395. Arch Microbiol 191, 903–911 (2009). https://doi.org/10.1007/s00203-009-0521-8

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