The substrate specificity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component

doi:10.1016/S0006-291X(02)02626-8

Biochemical and Biophysical Research Communications

Volume 299, Issue 2, 29 November 2002, Pages 247-251

https://doi.org/10.1016/S0006-291X(02)02626-8 Get rights and content

Abstract

The tripartite efflux systems MexAB–OprM and MexCD–OprJ of Pseudomonas aeruginosa each display characteristic substrate specificity against a variety of antimicrobial agents. The chimeric efflux system MexC–MexB–OprJ/ΔMexD constructed by exchange of MexD with MexB endowed the recombinant host the same resistance profile as MexAB–OprM rather than MexCD–OprJ. The change of substrate specificity was shown to be due to extrusion from the chimeric efflux system by cellular accumulation experiments using tetracycline, erythromycin, and ethidium bromide. Thus, we conclude that MexB and MexD are primary components of the efflux system responsible for sorting extrusion substrates.

Section snippets

Materials and methods

Bacteria and culture conditions. All P. aeruginosa strains used in this study are isogenic mutants of the laboratory strain PAO1 (prototroph). Bacterial cells were grown in L broth (1% wt/vol tryptone, 0.5% wt/vol yeast extract, and 0.5% wt/vol NaCl), L agar (L broth plus 1.5% wt/vol agar) or BM2 minimal medium [15] at 37 °C.

Construction of a host strain for characterization of efflux systems. Deletion or insertion mutagenesis of a target gene was performed using the previously established

Production of efflux components

Expression of the efflux systems in the P. aeruginosa strains were assessed using specific antibodies. Results are shown in Fig. 1. Deletion of mexD (in KG4514) (lane 3) from the MexC–MexD–OprJ-expressing strain KG2259 (lanes 1 and 2) resulted in a significant reduction in the level of detectable OprJ (lane 4), the product of the third gene of the mexC-mexD-oprJ efflux operon. This is presumably due to the polar effect caused by deletion of mexD. A similar phenomenon has been reported when the

Conclusion and comments

The substrate specificities of other efflux systems of P. aeruginosa other than MexAB–OprM and MexCD–OprJ have also been well characterized. Genes encoding RNDs, which are known to mediate resistance against antimicrobial agents, such as MexF [7] and MexY [6] from P. aeruginosa, AcrD [25] and AcrB [26] from Escherichia coli, and MtrD [27] from Neisseria gonorrhoeae were PCR-amplified and cloned into pTO001-His for expression in strain KG4514. Although the level of expression for all cloned

Acknowledgements

This research was supported by grants for scientific research to N.G. from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and from the Ministry of Health, Labor, and Welfare of Japan. T.M. and M.K. were recipients of a scholarship from the Japan Scholarship Foundation.

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^☆: Abbreviations: NFLX, norfloxacin; SPFX, sparfloxacin; CPR, cefpirome; CZOP, cefozopran; AZT, aztreonam; CBPC, carbenicillin; TC, tetracycline; CP, chloramphenicol; EM, erythromycin; EB, ethidium bromide; AF, acriflavine; ABM, MexAB–OprM; CDJ, MexCD–OprJ; CJ, MexC–ΔMexD–OprJ; CJ/D, MexC–ΔMexD–OprJ/MexD; CJ/B, MexC–ΔMexD–OprJ/MexB.

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The substrate specificity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component☆

Abstract

Section snippets

Materials and methods

Production of efflux components

Conclusion and comments

Acknowledgements

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

J. Struct. Biol.

Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine

Mol. Microbiol.

Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon

J. Bacteriol.

The outer membrane protein OprM of Pseudomonas aeruginosa is encoded by oprK of the mexA-mexB-oprK multidrug resistance operon

Antimicrob. Agents Chemother.

Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa

Mol. Microbiol.

Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides

Antimicrob. Agents Chemother.

Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa

Antimicrob. Agents Chemother.

Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa

Mol. Microbiol.

Substrate specificities of MexAB–OprM, MexCD–OprJ, and MexXY–OprM efflux pumps in Pseudomonas aeruginosa

Antimicrob. Agents Chemother.

Assignment of the substrate-selective subunits of the MexEF–OprN multidrug efflux pump of Pseudomonas aeruginosa

Antimicrob. Agents Chemother.

Inner membrane efflux components are responsible for β-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa

J. Bacteriol.