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. 1995 Aug;177(15):4557–4561. doi: 10.1128/jb.177.15.4557-4561.1995

Tetracycline/H+ antiport and Na+/H+ antiport catalyzed by the Bacillus subtilis TetA(L) transporter expressed in Escherichia coli.

A A Guffanti 1, T A Krulwich 1
PMCID: PMC177215  PMID: 7635843

Abstract

The properties of TetA(L)-dependent tetracycline/proton and Na+/proton antiport were studied in energized everted vesicles of Escherichia coli transformed with a cloned tetA(L) gene (pJTA1) from Bacillus subtilis. Inhibition patterns by valinomycin and nigericin indicated that both antiports were electrogenic, in contrast to the tetracycline/proton antiport encoded by gram-negative plasmid tet genes. Tetracycline uptake in the everted system was dependent upon a divalent cation, with cobalt being the preferred one. The apparent Km for tetracycline was markedly increased at pH 8.5 versus pH 7.5, whereas the Vmax was unchanged. The much higher apparent Km for Na+ decreased at pH 8.5 relative to that at pH 7.5, as did the Vmax. Na+ did not affect tetracycline uptake, nor did Co2+ and/or tetracycline affect Na+ uptake; complex patterns of inhibition by amiloride and analogs thereof were observed.

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Selected References

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  1. Amano H., Ives C. L., Bott K. F., Shishido K. A limited number of Bacillus subtilis strains carry a tetracycline-resistance determinant at a site close to the origin of replication. Biochim Biophys Acta. 1991 Feb 16;1088(2):251–258. doi: 10.1016/0167-4781(91)90061-p. [DOI] [PubMed] [Google Scholar]
  2. Goldberg E. B., Arbel T., Chen J., Karpel R., Mackie G. A., Schuldiner S., Padan E. Characterization of a Na+/H+ antiporter gene of Escherichia coli. Proc Natl Acad Sci U S A. 1987 May;84(9):2615–2619. doi: 10.1073/pnas.84.9.2615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Griffith J. K., Cuellar D. H., Fordyce C. A., Hutchings K. G., Mondragon A. A. Structure and function of the class C tetracycline/H+ antiporter: three independent groups of phenotypes are conferred by TetA (C). Mol Membr Biol. 1994 Oct-Dec;11(4):271–277. doi: 10.3109/09687689409160437. [DOI] [PubMed] [Google Scholar]
  4. Griffith J. K., Kogoma T., Corvo D. L., Anderson W. L., Kazim A. L. An N-terminal domain of the tetracycline resistance protein increases susceptibility to aminoglycosides and complements potassium uptake defects in Escherichia coli. J Bacteriol. 1988 Feb;170(2):598–604. doi: 10.1128/jb.170.2.598-604.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ives C. L., Bott K. F. Characterization of chromosomal DNA amplifications with associated tetracycline resistance in Bacillus subtilis. J Bacteriol. 1990 Sep;172(9):4936–4944. doi: 10.1128/jb.172.9.4936-4944.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ives C. L., Bott K. F. Cloned Bacillus subtilis chromosomal DNA mediates tetracycline resistance when present in multiple copies. J Bacteriol. 1989 Apr;171(4):1801–1810. doi: 10.1128/jb.171.4.1801-1810.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kleyman T. R., Cragoe E. J., Jr Amiloride and its analogs as tools in the study of ion transport. J Membr Biol. 1988 Oct;105(1):1–21. doi: 10.1007/BF01871102. [DOI] [PubMed] [Google Scholar]
  8. Kuroda T., Shimamoto T., Inaba K., Tsuda M., Tsuchiya T. Properties and sequence of the NhaA Na+/H+ antiporter of Vibrio parahaemolyticus. J Biochem. 1994 Nov;116(5):1030–1038. doi: 10.1093/oxfordjournals.jbchem.a124624. [DOI] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. 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]
  11. Levy S. B., McMurry L. M., Burdett V., Courvalin P., Hillen W., Roberts M. C., Taylor D. E. Nomenclature for tetracycline resistance determinants. Antimicrob Agents Chemother. 1989 Aug;33(8):1373–1374. doi: 10.1128/aac.33.8.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McMurry L. M., Park B. H., Burdett V., Levy S. B. Energy-dependent efflux mediated by class L (tetL) tetracycline resistance determinant from streptococci. Antimicrob Agents Chemother. 1987 Oct;31(10):1648–1650. doi: 10.1128/aac.31.10.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Neyfakh A. A., Bidnenko V. E., Chen L. B. Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4781–4785. doi: 10.1073/pnas.88.11.4781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Ogasawara N., Nakai S., Yoshikawa H. Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res. 1994;1(1):1–14. doi: 10.1093/dnares/1.1.1. [DOI] [PubMed] [Google Scholar]
  16. Padan E., Schuldiner S. Na+/H+ antiporters, molecular devices that couple the Na+ and H+ circulation in cells. J Bioenerg Biomembr. 1993 Dec;25(6):647–669. doi: 10.1007/BF00770252. [DOI] [PubMed] [Google Scholar]
  17. Pinner E., Kotler Y., Padan E., Schuldiner S. Physiological role of nhaB, a specific Na+/H+ antiporter in Escherichia coli. J Biol Chem. 1993 Jan 25;268(3):1729–1734. [PubMed] [Google Scholar]
  18. Pinner E., Padan E., Schuldiner S. Kinetic properties of NhaB, a Na+/H+ antiporter from Escherichia coli. J Biol Chem. 1994 Oct 21;269(42):26274–26279. [PubMed] [Google Scholar]
  19. Salyers A. A., Speer B. S., Shoemaker N. B. New perspectives in tetracycline resistance. Mol Microbiol. 1990 Jan;4(1):151–156. doi: 10.1111/j.1365-2958.1990.tb02025.x. [DOI] [PubMed] [Google Scholar]
  20. Speer B. S., Shoemaker N. B., Salyers A. A. Bacterial resistance to tetracycline: mechanisms, transfer, and clinical significance. Clin Microbiol Rev. 1992 Oct;5(4):387–399. doi: 10.1128/cmr.5.4.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Taglicht D., Padan E., Schuldiner S. Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli. J Biol Chem. 1991 Jun 15;266(17):11289–11294. [PubMed] [Google Scholar]
  22. Wilson C. R., Morgan A. E. Chromosomal-DNA amplification in Bacillus subtilis. J Bacteriol. 1985 Aug;163(2):445–453. doi: 10.1128/jb.163.2.445-453.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yamaguchi A., Ohmori H., Kaneko-Ohdera M., Nomura T., Sawai T. Delta pH-dependent accumulation of tetracycline in Escherichia coli. Antimicrob Agents Chemother. 1991 Jan;35(1):53–56. doi: 10.1128/aac.35.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]

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