Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Oct;175(19):6299–6313. doi: 10.1128/jb.175.19.6299-6313.1993

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

D Ma 1, D N Cook 1, M Alberti 1, N G Pon 1, H Nikaido 1, J E Hearst 1
PMCID: PMC206727  PMID: 8407802

Abstract

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.

Full text

PDF
6302

Images in this article

6306Image
on p.6306

Selected References

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

  1. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bouvier J., Richaud C., Higgins W., Bögler O., Stragier P. Cloning, characterization, and expression of the dapE gene of Escherichia coli. J Bacteriol. 1992 Aug;174(16):5265–5271. doi: 10.1128/jb.174.16.5265-5271.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Coleman W. G., Jr, Leive L. Two mutations which affect the barrier function of the Escherichia coli K-12 outer membrane. J Bacteriol. 1979 Sep;139(3):899–910. doi: 10.1128/jb.139.3.899-910.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cook D. N., Ma D., Pon N. G., Hearst J. E. Dynamics of DNA supercoiling by transcription in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10603–10607. doi: 10.1073/pnas.89.22.10603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DiNardo S., Voelkel K. A., Sternglanz R., Reynolds A. E., Wright A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell. 1982 Nov;31(1):43–51. doi: 10.1016/0092-8674(82)90403-2. [DOI] [PubMed] [Google Scholar]
  6. Dorman C. J., Lynch A. S., Ni Bhriain N., Higgins C. F. DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus. Mol Microbiol. 1989 Apr;3(4):531–540. doi: 10.1111/j.1365-2958.1989.tb00199.x. [DOI] [PubMed] [Google Scholar]
  7. Gamper H. B., Hearst J. E. A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes. Cell. 1982 May;29(1):81–90. doi: 10.1016/0092-8674(82)90092-7. [DOI] [PubMed] [Google Scholar]
  8. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ghosal D., Sommer H., Saedler H. Nucleotide sequence of the transposable DNA-element IS2. Nucleic Acids Res. 1979 Mar;6(3):1111–1122. doi: 10.1093/nar/6.3.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hayashi S., Wu H. C. Lipoproteins in bacteria. J Bioenerg Biomembr. 1990 Jun;22(3):451–471. doi: 10.1007/BF00763177. [DOI] [PubMed] [Google Scholar]
  11. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  12. Jaurin B., Normark S. Insertion of IS2 creates a novel ampC promoter in Escherichia coli. Cell. 1983 Mar;32(3):809–816. doi: 10.1016/0092-8674(83)90067-3. [DOI] [PubMed] [Google Scholar]
  13. Kirkegaard K., Wang J. C. Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop. J Mol Biol. 1985 Oct 5;185(3):625–637. doi: 10.1016/0022-2836(85)90075-0. [DOI] [PubMed] [Google Scholar]
  14. Klein J. R., Henrich B., Plapp R. Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli. Mol Gen Genet. 1991 Nov;230(1-2):230–240. doi: 10.1007/BF00290673. [DOI] [PubMed] [Google Scholar]
  15. Klein J. R., Plapp R. Locations of the envCD genes on the physical map of the Escherichia coli chromosome. J Bacteriol. 1992 Jun;174(11):3828–3829. doi: 10.1128/jb.174.11.3828-3829.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  17. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  18. Leive L., Telesetsky S., Coleman W. G., Jr, Carr D. Tetracyclines of various hydrophobicities as a probe for permeability of Escherichia coli outer membranes. Antimicrob Agents Chemother. 1984 May;25(5):539–544. doi: 10.1128/aac.25.5.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lynch A. S., Wang J. C. Anchoring of DNA to the bacterial cytoplasmic membrane through cotranscriptional synthesis of polypeptides encoding membrane proteins or proteins for export: a mechanism of plasmid hypernegative supercoiling in mutants deficient in DNA topoisomerase I. J Bacteriol. 1993 Mar;175(6):1645–1655. doi: 10.1128/jb.175.6.1645-1655.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Manoil C., Mekalanos J. J., Beckwith J. Alkaline phosphatase fusions: sensors of subcellular location. J Bacteriol. 1990 Feb;172(2):515–518. doi: 10.1128/jb.172.2.515-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Menzel R., Gellert M. Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell. 1983 Aug;34(1):105–113. doi: 10.1016/0092-8674(83)90140-x. [DOI] [PubMed] [Google Scholar]
  25. Michel G., Di Savino D., Starka J. Phospholipid composition and phenotypic correction of an envC division mutant of Escherichia coli. J Bacteriol. 1977 Jan;129(1):145–150. doi: 10.1128/jb.129.1.145-150.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakamura H. Gene-Controlled Resistance to Acriflavine and Other Basic Dyes in Escherichia coli. J Bacteriol. 1965 Jul;90(1):8–14. doi: 10.1128/jb.90.1.8-14.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nakamura H. Genetic determination of resistance to acriflavine, phenethyl alcohol, and sodium dodecyl sulfate in Escherichia coli. J Bacteriol. 1968 Oct;96(4):987–996. doi: 10.1128/jb.96.4.987-996.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nakamura H. Plasmid-instability in acrA mutants of Escherichia coli K12. J Gen Microbiol. 1974 Sep;84(1):85–93. doi: 10.1099/00221287-84-1-85. [DOI] [PubMed] [Google Scholar]
  29. Nakamura H., Suganuma A. Membrane mutation associated with sensitivity to acriflavine in Escherichia coli. J Bacteriol. 1972 Apr;110(1):329–335. doi: 10.1128/jb.110.1.329-335.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakamura H., Tojo T., Greenberg J. Interaction of the expression of two membrane genes, acrA and plsA, in Escherichia coli K-12. J Bacteriol. 1975 Jun;122(3):874–879. doi: 10.1128/jb.122.3.874-879.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nies D. H., Nies A., Chu L., Silver S. Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7351–7355. doi: 10.1073/pnas.86.19.7351. [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. 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]
  34. Pruss G. J., Manes S. H., Drlica K. Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell. 1982 Nov;31(1):35–42. doi: 10.1016/0092-8674(82)90402-0. [DOI] [PubMed] [Google Scholar]
  35. Raji A., Zabel D. J., Laufer C. S., Depew R. E. Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I. J Bacteriol. 1985 Jun;162(3):1173–1179. doi: 10.1128/jb.162.3.1173-1179.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rudd K. E., Miller W., Ostell J., Benson D. A. Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map. Nucleic Acids Res. 1990 Jan 25;18(2):313–321. doi: 10.1093/nar/18.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Seiffer D., Klein J. R., Plapp R. EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli. FEMS Microbiol Lett. 1993 Mar 1;107(2-3):175–178. doi: 10.1111/j.1574-6968.1993.tb06026.x. [DOI] [PubMed] [Google Scholar]
  38. Stárková Z., Bonnaveiro N., Stárka J. Hydrolysis of phospholipids by phospholipase C in intact cells of wild-type and envelope mutants of Escherichia coli K12. FEBS Lett. 1981 Aug 3;130(2):261–264. doi: 10.1016/0014-5793(81)81134-9. [DOI] [PubMed] [Google Scholar]
  39. Yamaguchi K., Yu F., Inouye M. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell. 1988 May 6;53(3):423–432. doi: 10.1016/0092-8674(88)90162-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES