Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Feb;177(4):998–1007. doi: 10.1128/jb.177.4.998-1007.1995

Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

D G Thanassi 1, G S Suh 1, H Nikaido 1
PMCID: PMC176695  PMID: 7860612

Abstract

Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented.

Full Text

The Full Text of this article is available as a PDF (354.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ALBERT A., REES C. W. Avidity of the tetracyclines for the cations of metals. Nature. 1956 Mar 3;177(4505):433–434. doi: 10.1038/177433a0. [DOI] [PubMed] [Google Scholar]
  2. Argast M., Beck C. F. Tetracycline uptake by susceptible Escherichia coli cells. Arch Microbiol. 1985 Apr;141(3):260–265. doi: 10.1007/BF00408069. [DOI] [PubMed] [Google Scholar]
  3. Bavoil P., Nikaido H., von Meyenburg K. Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein. Mol Gen Genet. 1977 Dec 14;158(1):23–33. doi: 10.1007/BF00455116. [DOI] [PubMed] [Google Scholar]
  4. Bayer M. H., Costello G. P., Bayer M. E. Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites. J Bacteriol. 1982 Feb;149(2):758–767. doi: 10.1128/jb.149.2.758-767.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chopra I., Hawkey P. M., Hinton M. Tetracyclines, molecular and clinical aspects. J Antimicrob Chemother. 1992 Mar;29(3):245–277. doi: 10.1093/jac/29.3.245. [DOI] [PubMed] [Google Scholar]
  6. Cohen S. P., Hächler H., Levy S. B. Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli. J Bacteriol. 1993 Mar;175(5):1484–1492. doi: 10.1128/jb.175.5.1484-1492.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen S. P., McMurry L. M., Levy S. B. marA locus causes decreased expression of OmpF porin in multiple-antibiotic-resistant (Mar) mutants of Escherichia coli. J Bacteriol. 1988 Dec;170(12):5416–5422. doi: 10.1128/jb.170.12.5416-5422.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dinh T., Paulsen I. T., Saier M. H., Jr A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria. J Bacteriol. 1994 Jul;176(13):3825–3831. doi: 10.1128/jb.176.13.3825-3831.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eckert B., Beck C. F. Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli. J Biol Chem. 1989 Jul 15;264(20):11663–11670. [PubMed] [Google Scholar]
  10. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. George A. M., Levy S. B. Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline. J Bacteriol. 1983 Aug;155(2):531–540. doi: 10.1128/jb.155.2.531-540.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heller K. B., Lin E. C., Wilson T. H. Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli. J Bacteriol. 1980 Oct;144(1):274–278. doi: 10.1128/jb.144.1.274-278.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ishidate K., Creeger E. S., Zrike J., Deb S., Glauner B., MacAlister T. J., Rothfield L. I. Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope. J Biol Chem. 1986 Jan 5;261(1):428–443. [PubMed] [Google Scholar]
  14. Jacoby G. H., Young K. D. Unequal distribution of penicillin-binding proteins among inner membrane vesicles of Escherichia coli. J Bacteriol. 1988 Aug;170(8):3660–3667. doi: 10.1128/jb.170.8.3660-3667.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jogun K. H., Stezowski J. J. Chemical-structural properties of tetracycline derivatives. 2. Coordination and conformational aspects of oxytetracycline metal ion complexation. J Am Chem Soc. 1976 Sep 15;98(19):6018–6026. doi: 10.1021/ja00435a040. [DOI] [PubMed] [Google Scholar]
  16. Kashket E. R. Stoichiometry of the H+-ATPase of growing and resting, aerobic Escherichia coli. Biochemistry. 1982 Oct 26;21(22):5534–5538. doi: 10.1021/bi00265a024. [DOI] [PubMed] [Google Scholar]
  17. Levy S. B. Active efflux mechanisms for antimicrobial resistance. Antimicrob Agents Chemother. 1992 Apr;36(4):695–703. doi: 10.1128/aac.36.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li X. Z., Livermore D. M., Nikaido H. Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrob Agents Chemother. 1994 Aug;38(8):1732–1741. doi: 10.1128/aac.38.8.1732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lomovskaya O., Lewis K. Emr, an Escherichia coli locus for multidrug resistance. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8938–8942. doi: 10.1073/pnas.89.19.8938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ma D., Cook D. N., Alberti M., Pon N. G., Nikaido H., Hearst J. E. Molecular cloning and characterization of acrA and acrE genes of Escherichia coli. J Bacteriol. 1993 Oct;175(19):6299–6313. doi: 10.1128/jb.175.19.6299-6313.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McMurry L. M., Aronson D. A., Levy S. B. Susceptible Escherichia coli cells can actively excrete tetracyclines. Antimicrob Agents Chemother. 1983 Oct;24(4):544–551. doi: 10.1128/aac.24.4.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McMurry L. M., Cullinane J. C., Levy S. B. Transport of the lipophilic analog minocycline differs from that of tetracycline in susceptible and resistant Escherichia coli strains. Antimicrob Agents Chemother. 1982 Nov;22(5):791–799. doi: 10.1128/aac.22.5.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McMurry L., Levy S. B. Two transport systems for tetracycline in sensitive Escherichia coli: critical role for an initial rapid uptake system insensitive to energy inhibitors. Antimicrob Agents Chemother. 1978 Aug;14(2):201–209. doi: 10.1128/aac.14.2.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McMurry L., Petrucci R. E., Jr, Levy S. B. Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3974–3977. doi: 10.1073/pnas.77.7.3974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mühlradt P. F., Menzel J., Golecki J. R., Speth V. Lateral mobility and surface density of lipopolysaccharide in the outer membrane of Salmonella typhimurium. Eur J Biochem. 1974 Apr 16;43(3):533–539. doi: 10.1111/j.1432-1033.1974.tb03440.x. [DOI] [PubMed] [Google Scholar]
  27. Nikaido H. Outer membrane of Salmonella typhimurium. Transmembrane diffusion of some hydrophobic substances. Biochim Biophys Acta. 1976 Apr 16;433(1):118–132. doi: 10.1016/0005-2736(76)90182-6. [DOI] [PubMed] [Google Scholar]
  28. Nikaido H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science. 1994 Apr 15;264(5157):382–388. doi: 10.1126/science.8153625. [DOI] [PubMed] [Google Scholar]
  29. Nikaido H., Rosenberg E. Y. Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli. J Gen Physiol. 1981 Feb;77(2):121–135. doi: 10.1085/jgp.77.2.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nikaido H., Rosenberg E. Y., Foulds J. Porin channels in Escherichia coli: studies with beta-lactams in intact cells. J Bacteriol. 1983 Jan;153(1):232–240. doi: 10.1128/jb.153.1.232-240.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nikaido H., Thanassi D. G. Penetration of lipophilic agents with multiple protonation sites into bacterial cells: tetracyclines and fluoroquinolones as examples. Antimicrob Agents Chemother. 1993 Jul;37(7):1393–1399. doi: 10.1128/aac.37.7.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  34. Plésiat P., Nikaido H. Outer membranes of gram-negative bacteria are permeable to steroid probes. Mol Microbiol. 1992 May;6(10):1323–1333. doi: 10.1111/j.1365-2958.1992.tb00853.x. [DOI] [PubMed] [Google Scholar]
  35. Poole K., Krebes K., McNally C., Neshat S. Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J Bacteriol. 1993 Nov;175(22):7363–7372. doi: 10.1128/jb.175.22.7363-7372.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pugsley A. P., Schnaitman C. A. Outer membrane proteins of Escherichia coli. VII. Evidence that bacteriophage-directed protein 2 functions as a pore. J Bacteriol. 1978 Mar;133(3):1181–1189. doi: 10.1128/jb.133.3.1181-1189.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. RIGLER N. E., BAG S. P., LEYDEN D. E., SUDMEIER J. L., REILLEY C. N. DETERMINATION OF A PROTONATION SCHEME OF TETRACYCLINE USING NUCLEAR MAGNETIC RESONANCE. Anal Chem. 1965 Jun;37:872–875. doi: 10.1021/ac60226a022. [DOI] [PubMed] [Google Scholar]
  38. Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sen K., Hellman J., Nikaido H. Porin channels in intact cells of Escherichia coli are not affected by Donnan potentials across the outer membrane. J Biol Chem. 1988 Jan 25;263(3):1182–1187. [PubMed] [Google Scholar]
  40. Smit J., Kamio Y., Nikaido H. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J Bacteriol. 1975 Nov;124(2):942–958. doi: 10.1128/jb.124.2.942-958.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  42. Stock J. B., Rauch B., Roseman S. Periplasmic space in Salmonella typhimurium and Escherichia coli. J Biol Chem. 1977 Nov 10;252(21):7850–7861. [PubMed] [Google Scholar]
  43. Sugawara E., Nikaido H. Pore-forming activity of OmpA protein of Escherichia coli. J Biol Chem. 1992 Feb 5;267(4):2507–2511. [PubMed] [Google Scholar]
  44. Witholt B., Boekhout M., Brock M., Kingma J., Heerikhuizen H. V., Leij L. D. An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal Biochem. 1976 Jul;74(1):160–170. doi: 10.1016/0003-2697(76)90320-1. [DOI] [PubMed] [Google Scholar]
  45. Yamaguchi A., Adachi K., Sawai T. Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli. FEBS Lett. 1990 Jun 4;265(1-2):17–19. doi: 10.1016/0014-5793(90)80872-g. [DOI] [PubMed] [Google Scholar]
  46. Yamaguchi A., Iwasaki-Ohba Y., Ono N., Kaneko-Ohdera M., Sawai T. Stoichiometry of metal-tetracycline/H+ antiport mediated by transposon Tn10-encoded tetracycline resistance protein in Escherichia coli. FEBS Lett. 1991 May 6;282(2):415–418. doi: 10.1016/0014-5793(91)80527-a. [DOI] [PubMed] [Google Scholar]
  47. Yamaguchi A., Udagawa T., Sawai T. Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein. J Biol Chem. 1990 Mar 25;265(9):4809–4813. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES