Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1997 Dec;41(12):2699–2704. doi: 10.1128/aac.41.12.2699

Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

D G White 1, K Maneewannakul 1, E von Hofe 1, M Zillman 1, W Eisenberg 1, A K Field 1, S B Levy 1
PMCID: PMC164191  PMID: 9420041

Abstract

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant. Two antisense phosphorothioate oligonucleotides, one targeted to marO and the other targeted to marA of the mar operon, introduced by heat shock or electroporation reduced LacZ expression in the strain having the marA::lacZ fusion. One antisense oligonucleotide, tested against a Mar mutant of E. coli ML308-225, increased the bactericidal activity of norfloxacin. These studies demonstrate the efficacy of exogenously delivered antisense oligonucleotides targeted to the marRAB operon in inhibiting expression of this chromosomal regulatory locus.

Full Text

The Full Text of this article is available as a PDF (453.1 KB).

Selected References

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

  1. Alekshun M. N., Levy S. B. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother. 1997 Oct;41(10):2067–2075. doi: 10.1128/aac.41.10.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ariza R. R., Cohen S. P., Bachhawat N., Levy S. B., Demple B. Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli. J Bacteriol. 1994 Jan;176(1):143–148. doi: 10.1128/jb.176.1.143-148.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Asako H., Nakajima H., Kobayashi K., Kobayashi M., Aono R. Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl Environ Microbiol. 1997 Apr;63(4):1428–1433. doi: 10.1128/aem.63.4.1428-1433.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Cohen S. P., Levy S. B., Foulds J., Rosner J. L. Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway. J Bacteriol. 1993 Dec;175(24):7856–7862. doi: 10.1128/jb.175.24.7856-7862.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen S. P., McMurry L. M., Hooper D. C., Wolfson J. S., Levy S. B. Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction. Antimicrob Agents Chemother. 1989 Aug;33(8):1318–1325. doi: 10.1128/aac.33.8.1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen S. P., Yan W., Levy S. B. A multidrug resistance regulatory chromosomal locus is widespread among enteric bacteria. J Infect Dis. 1993 Aug;168(2):484–488. doi: 10.1093/infdis/168.2.484. [DOI] [PubMed] [Google Scholar]
  8. Gambino L., Gracheck S. J., Miller P. F. Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli. J Bacteriol. 1993 May;175(10):2888–2894. doi: 10.1128/jb.175.10.2888-2894.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasparro F. P., Edelson R. L., O'Malley M. E., Ugent S. J., Wong H. H. Photoactivatable antisense DNA: suppression of ampicillin resistance in normally resistant Escherichia coli. Antisense Res Dev. 1991 Summer;1(2):117–140. doi: 10.1089/ard.1991.1.117. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. George A. M., Levy S. B. Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics. J Bacteriol. 1983 Aug;155(2):541–548. doi: 10.1128/jb.155.2.541-548.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldman J. D., White D. G., Levy S. B. Multiple antibiotic resistance (mar) locus protects Escherichia coli from rapid cell killing by fluoroquinolones. Antimicrob Agents Chemother. 1996 May;40(5):1266–1269. doi: 10.1128/aac.40.5.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ho S. P., Britton D. H., Stone B. A., Behrens D. L., Leffet L. M., Hobbs F. W., Miller J. A., Trainor G. L. Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries. Nucleic Acids Res. 1996 May 15;24(10):1901–1907. doi: 10.1093/nar/24.10.1901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hächler H., Cohen S. P., Levy S. B. marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli. J Bacteriol. 1991 Sep;173(17):5532–5538. doi: 10.1128/jb.173.17.5532-5538.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jair K. W., Martin R. G., Rosner J. L., Fujita N., Ishihama A., Wolf R. E., Jr Purification and regulatory properties of MarA protein, a transcriptional activator of Escherichia coli multiple antibiotic and superoxide resistance promoters. J Bacteriol. 1995 Dec;177(24):7100–7104. doi: 10.1128/jb.177.24.7100-7104.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jayaraman K., McParland K., Miller P., Ts'o P. O. Selective inhibition of Escherichia coli protein synthesis and growth by nonionic oligonucleotides complementary to the 3' end of 16S rRNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1537–1541. doi: 10.1073/pnas.78.3.1537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maneewannakul K., Levy S. B. Identification for mar mutants among quinolone-resistant clinical isolates of Escherichia coli. Antimicrob Agents Chemother. 1996 Jul;40(7):1695–1698. doi: 10.1128/aac.40.7.1695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McMurry L. M., George A. M., Levy S. B. Active efflux of chloramphenicol in susceptible Escherichia coli strains and in multiple-antibiotic-resistant (Mar) mutants. Antimicrob Agents Chemother. 1994 Mar;38(3):542–546. doi: 10.1128/aac.38.3.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Neu H. C. The crisis in antibiotic resistance. Science. 1992 Aug 21;257(5073):1064–1073. doi: 10.1126/science.257.5073.1064. [DOI] [PubMed] [Google Scholar]
  21. Okusu H., Ma D., Nikaido H. AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J Bacteriol. 1996 Jan;178(1):306–308. doi: 10.1128/jb.178.1.306-308.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pari G. S., Field A. K., Smith J. A. Potent antiviral activity of an antisense oligonucleotide complementary to the intron-exon boundary of human cytomegalovirus genes UL36 and UL37. Antimicrob Agents Chemother. 1995 May;39(5):1157–1161. doi: 10.1128/aac.39.5.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rahman M. A., Summerton J., Foster E., Cunningham K., Stirchak E., Weller D., Schaup H. W. Antibacterial activity and inhibition of protein synthesis in Escherichia coli by antisense DNA analogs. Antisense Res Dev. 1991 Winter;1(4):319–327. doi: 10.1089/ard.1991.1.319. [DOI] [PubMed] [Google Scholar]
  24. Ramazeilles C., Mishra R. K., Moreau S., Pascolo E., Toulmé J. J. Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7859–7863. doi: 10.1073/pnas.91.17.7859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rapaport E., Levina A., Metelev V., Zamecnik P. C. Antimycobacterial activities of antisense oligodeoxynucleotide phosphorothioates in drug-resistant strains. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):709–713. doi: 10.1073/pnas.93.2.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rapaport E., Misiura K., Agrawal S., Zamecnik P. Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8577–8580. doi: 10.1073/pnas.89.18.8577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Seoane A. S., Levy S. B. Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli. J Bacteriol. 1995 Jun;177(12):3414–3419. doi: 10.1128/jb.177.12.3414-3419.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seoane A. S., Levy S. B. Identification of new genes regulated by the marRAB operon in Escherichia coli. J Bacteriol. 1995 Feb;177(3):530–535. doi: 10.1128/jb.177.3.530-535.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wagner R. W. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994 Nov 24;372(6504):333–335. doi: 10.1038/372333a0. [DOI] [PubMed] [Google Scholar]
  31. White D. G., Goldman J. D., Demple B., Levy S. B. Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli. J Bacteriol. 1997 Oct;179(19):6122–6126. doi: 10.1128/jb.179.19.6122-6126.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES