The authors previously described , an operon involved in proton-dependent solvent efflux in the solvent-tolerant S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in S12 but is solely involved in multidrug resistance.


Article metrics loading...

Loading full text...

Full text loading...



  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990). Basic local alignment search tool. J Mol Biol 215, 403-410.[CrossRef] [Google Scholar]
  2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (1991).Current Protocols in Molecular Biology. New York: Greene Publishing Associates.
  3. Birnboim, H. C. & Doly, J.(1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523.[CrossRef] [Google Scholar]
  4. Dennis, J. J. & Sokol, P. A.(1995). Electrotransformation of Pseudomonas. Methods Mol Biol 47, 125-133. [Google Scholar]
  5. Dennis, J. J. & Zylstra, G. J.(1998). Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of Gram-negative bacterial genomes. Appl Environ Microbiol 64, 2710-2715. [Google Scholar]
  6. Dinh, T., Paulsen, I. T. & Saier, M. H.Jr(1994). A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria. J Bacteriol 176, 3825-3831. [Google Scholar]
  7. Fukumori, F., Hirayama, H., Takami, H., Inoue, A. & Horikoshi, K.(1998). Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent tolerance. Extremophiles 2, 395-400.[CrossRef] [Google Scholar]
  8. Guan, L., Ehrmann, M., Yoneyma, H. & Nakae, T.(1999). Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexAB-OprM extrusion pump in Pseudomonas aeruginosa. J Biol Chem 274, 10517-10522.[CrossRef] [Google Scholar]
  9. Hartmans, S., Smits, J. P., van der Werf, M., Volkering, J. F. & de Bont, J. A. M.(1989). Metabolism of styrene oxide and 2-phenylethanol in the styrene degrading Xanthobacter strain 124X. Appl Environ Microbiol 55, 2850-2855. [Google Scholar]
  10. Isken, S. & de Bont, J. A. M.(1996). Active efflux of toluene in a solvent-resistant bacterium. J Bacteriol 178, 6056-6058. [Google Scholar]
  11. Isken, S. & de Bont, J. A. M.(1998). Bacteria tolerant to organic solvents. Extremophiles 2, 229-238.[CrossRef] [Google Scholar]
  12. Kieboom, J. & de Bont, J. A. M.(2000). Mechanisms of organic solvent resistance in bacteria. In Bacterial Stress Responses , pp. 393-402. Edited by G. Storz & R. Hengge-Aronis. Washington, DC:American Society for Microbiology.
  13. Kieboom, J., Dennis, J. J., de Bont, J. A. M. & Zylstra, G. J.(1998a). Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance. J Biol Chem 273, 85-91.[CrossRef] [Google Scholar]
  14. Kieboom, J., Dennis, J. J., Zylstra, G. J. & de Bont, J. A. M.(1998b). Active efflux of organic solvents in Pseudomonas putida S12 is induced by solvents. J Bacteriol 180, 6769-6772. [Google Scholar]
  15. Kim, K., Lee, L., Lee, K. & Lim, D.(1998). Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73. J Bacteriol 180, 3692-3696. [Google Scholar]
  16. Kohler, T., Michea-Hamzehpour, M., Henze, U., Gotoh, N., Curty, L. K. & Pechere, J.-C.(1997). Characterization of the MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol Microbiol 23, 345-354.[CrossRef] [Google Scholar]
  17. Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M.II & Peterson, K. M.(1994). pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16, 801-802. [Google Scholar]
  18. Li, X.-Z. & Poole, K.(1999). Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance. Can J Microbiol 45, 18-22.[CrossRef] [Google Scholar]
  19. Li, X.-Z., Zhang, L. & Poole, K.(1998). Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance. J Bacteriol 180, 2987-2991. [Google Scholar]
  20. Maniatis, T. E., Fritsch, E. F. & Sambrook, J. (1982).Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  21. Miller, J. (1972).Experiments in Molecular Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  22. Mosqueda, G. & Ramos, J. L.(2000). A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism. J Bacteriol 182, 937-43.[CrossRef] [Google Scholar]
  23. Nikaido, H.(1994). Prevention of drug access to bacterial targets: role of permeability barriers and active efflux. Science 264, 382-388.[CrossRef] [Google Scholar]
  24. Nikaido, H.(1996). Multidrug efflux pumps of Gram-negative bacteria. J Bacteriol 178, 5853-5859. [Google Scholar]
  25. Paulsen, I. T., Brown, M. H. & Skurray, R. A.(1996). Proton-dependent multidrug efflux systems. Microbiol Rev 60, 575-608. [Google Scholar]
  26. Poole, K., Krebes, K., McNally, C. & Neshat, S.(1993). Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J Bacteriol 175, 7363-7372. [Google Scholar]
  27. Poole, K., Tetro, K., Zhao, Q., Neshat, S., Heinrichs, D. E. & Bianco, N.(1996). Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression. Antimicrob Agents Chemother 40, 2021-2028. [Google Scholar]
  28. Ramos, J. L., Duque, E., Godoy, P. & Segura, A.(1998). Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E. J Bacteriol 180, 3323-3329. [Google Scholar]
  29. Rothmel, R. K., Shinabarger, D. L., Parsek, M. R., Aldrich, T. L. & Chakrabarty, A. M.(1991). Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting. J Bacteriol 173, 4717-4724. [Google Scholar]
  30. Saier, M. H.Jr, Tam, R., Reizer, A. & Reizer, J.(1994). Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Mol Microbiol 11, 841-847.[CrossRef] [Google Scholar]
  31. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  32. Vogelstein, B. & Gillespie, D.(1979). Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci USA 76, 615-619.[CrossRef] [Google Scholar]
  33. Weber, F. J. & de Bont, J. A. M.(1996). Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes. Biochim Biophys Acta 1286, 225-245.[CrossRef] [Google Scholar]
  34. Weber, F. J., Ooijkaas, L. P., Schemen, R. M. W., Hartmans, S. & de Bont, J. A. M.(1993). Adaptation of Pseudomonas putida S12 to high concentrations of styrene and other organic solvents. Appl Environ Microbiol 59, 3502-3504. [Google Scholar]
  35. West, S. E., Schweizer, H. P., Dall, C., Sample, A. K. & Runyen-Janecky, L. J.(1994). Construction of improved Escherichia–Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148, 81-86.[CrossRef] [Google Scholar]
  36. Westbrock-Wadman, S., Sherman, D. R., Hickey & 7 other authors (1999). Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrob Agents Chemother 43, 2975–83. [Google Scholar]
  37. Yanisch-Perron, C., Vieira, J. & Messing, J.(1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error