Abstract
To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide. Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium. The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments. Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E. coli. Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules. Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate. These observations are indicative for a drug-proton antiport system. A lmrP deletion mutant was constructed via homologous recombinant using DNA fragments of the flanking region of the gene. The L. lactis (delta lmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of the functional drug-proton anti-porter LmrP, L. lactis is able to overexpress another, ATP-dependent, drug extrusion system. These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N. (1994) J. Bacteriol. 176, 6957-6964).
Highlights
For many years antibiotics have been found effective in the treatment of several infectious diseases caused by various pathogens
Cloning of the Lactococcal Drug Resistance Determinant lmrP—It has been shown that L. lactis MG1363 contains at least two different transport systems that are involved in efflux-mediated multidrug resistance (Bolhuis et al, 1994)
The DNA library was transformed to E. coli HB101, which does not grow on solid media containing ethidium bromide at concentrations above 75 g/ml
Summary
For many years antibiotics have been found effective in the treatment of several infectious diseases caused by various pathogens. One of the mechanisms underlying antibiotic resistance involves the extrusion of the compounds by an efflux pump or carrier (Tennent et al, 1989; Levi, 1992; Bolhuis et al, 1994; The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X89779. The most intriguing mechanisms of drug extrusion are those that can handle a wide variety of structurally unrelated compounds (antibiotics, drugs, etc.), and those are often referred to as multidrug resistance (MDR) transporters. The bacterial MDR type transporters as well as several closely related specific drug extrusion systems (SDR) can be divided into four groups on the basis of their relatedness in the primary sequences, similarity in the global molecular structure, and/or mechanism of energy coupling (Nikaido, 1994). The third group of secondary drug extrusion transporters is formed by the resistance-nodulation division (RND) family, found in Gram-negative bacteria (Saier et al, 1994). We describe the gene cloning, characterization, mutant construction, and functional analysis of LmrP
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
