Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Mar;175(6):1645–1655. doi: 10.1128/jb.175.6.1645-1655.1993

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.

A S Lynch 1, J C Wang 1
PMCID: PMC203958  PMID: 8383663

Abstract

A homologous set of plasmids expressing tet, lacY, and melB, genes encoding integral cytoplasmic membrane proteins, and tolC and ampC, genes encoding proteins for export through the cytoplasmic membrane, was constructed for studying the effects of transcription and translation of such genes on the hypernegative supercoiling of plasmids in Escherichia coli cells deficient in DNA topoisomerase I. The results support the view that intracellular bacterial DNA is anchored to the cytoplasmic membrane at many points through cotranscriptional synthesis of membrane proteins or proteins designated for export across the cytoplasmic membrane; in the latter case, the presence of the signal peptide appears to be unnecessary for cotranscriptional membrane association.

Full text

PDF
1645

Images in this article

1650Image
on p.1650
1650Image
on p.1650
1651Image
on p.1651
1651Image
on p.1651
1652Image
on p.1652

Selected References

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

  1. Ahrem B., Hoffschulte H. K., Müller M. In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation. J Cell Biol. 1989 May;108(5):1637–1646. doi: 10.1083/jcb.108.5.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balbás P., Soberón X., Merino E., Zurita M., Lomeli H., Valle F., Flores N., Bolivar F. Plasmid vector pBR322 and its special-purpose derivatives--a review. Gene. 1986;50(1-3):3–40. doi: 10.1016/0378-1119(86)90307-0. [DOI] [PubMed] [Google Scholar]
  3. Bibi E., Kaback H. R. In vivo expression of the lacY gene in two segments leads to functional lac permease. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4325–4329. doi: 10.1073/pnas.87.11.4325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bieker K. L., Silhavy T. J. PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli. Cell. 1990 Jun 1;61(5):833–842. doi: 10.1016/0092-8674(90)90193-i. [DOI] [PubMed] [Google Scholar]
  5. Botfield M. C., Naguchi K., Tsuchiya T., Wilson T. H. Membrane topology of the melibiose carrier of Escherichia coli. J Biol Chem. 1992 Jan 25;267(3):1818–1822. [PubMed] [Google Scholar]
  6. Calamia J., Manoil C. lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Dürrenberger M., Bjornsti M. A., Uetz T., Hobot J. A., Kellenberger E. Intracellular location of the histonelike protein HU in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4757–4768. doi: 10.1128/jb.170.10.4757-4768.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Fichot O., Girard M. An improved method for sequencing of RNA templates. Nucleic Acids Res. 1990 Oct 25;18(20):6162–6162. doi: 10.1093/nar/18.20.6162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Franco R. J., Drlica K. Gyrase inhibitors can increase gyrA expression and DNA supercoiling. J Bacteriol. 1989 Dec;171(12):6573–6579. doi: 10.1128/jb.171.12.6573-6579.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Guillerez J., Gazeau M., Dreyfus M. In the Escherichia coli lacZ gene the spacing between the translating ribosomes is insensitive to the efficiency of translation initiation. Nucleic Acids Res. 1991 Dec 25;19(24):6743–6750. doi: 10.1093/nar/19.24.6743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hackett J., Reeves P. Primary structure of the tolC gene that codes for an outer membrane protein of Escherichia coli K12. Nucleic Acids Res. 1983 Sep 24;11(18):6487–6495. doi: 10.1093/nar/11.18.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hickman R. K., McMurry L. M., Levy S. B. Overproduction and purification of the Tn10-specified inner membrane tetracycline resistance protein Tet using fusions to beta-galactosidase. Mol Microbiol. 1990 Aug;4(8):1241–1251. doi: 10.1111/j.1365-2958.1990.tb00703.x. [DOI] [PubMed] [Google Scholar]
  19. Hiraga S., Niki H., Ogura T., Ichinose C., Mori H., Ezaki B., Jaffé A. Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells. J Bacteriol. 1989 Mar;171(3):1496–1505. doi: 10.1128/jb.171.3.1496-1505.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ito K., Akiyama Y. In vivo analysis of integration of membrane proteins in Escherichia coli. Mol Microbiol. 1991 Sep;5(9):2243–2253. doi: 10.1111/j.1365-2958.1991.tb02154.x. [DOI] [PubMed] [Google Scholar]
  21. Jaurin B., Grundström T. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of beta-lactamases of the penicillinase type. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4897–4901. doi: 10.1073/pnas.78.8.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Josefsson L. G., Randall L. L. Different exported proteins in E. coli show differences in the temporal mode of processing in vivo. Cell. 1981 Jul;25(1):151–157. doi: 10.1016/0092-8674(81)90239-7. [DOI] [PubMed] [Google Scholar]
  23. Kammerer W., Deuschle U., Gentz R., Bujard H. Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process. EMBO J. 1986 Nov;5(11):2995–3000. doi: 10.1002/j.1460-2075.1986.tb04597.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Koshland D., Botstein D. Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane. Cell. 1982 Oct;30(3):893–902. doi: 10.1016/0092-8674(82)90294-x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Lockshon D., Morris D. R. Positively supercoiled plasmid DNA is produced by treatment of Escherichia coli with DNA gyrase inhibitors. Nucleic Acids Res. 1983 May 25;11(10):2999–3017. doi: 10.1093/nar/11.10.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lodge J. K., Kazic T., Berg D. E. Formation of supercoiling domains in plasmid pBR322. J Bacteriol. 1989 Apr;171(4):2181–2187. doi: 10.1128/jb.171.4.2181-2187.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Masukata H., Tomizawa J. A mechanism of formation of a persistent hybrid between elongating RNA and template DNA. Cell. 1990 Jul 27;62(2):331–338. doi: 10.1016/0092-8674(90)90370-t. [DOI] [PubMed] [Google Scholar]
  29. Niki H., Imamura R., Ogura T., Hiraga S. Nucleotide sequence of the tolC gene of Escherichia coli. Nucleic Acids Res. 1990 Sep 25;18(18):5547–5547. doi: 10.1093/nar/18.18.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ostrander E. A., Benedetti P., Wang J. C. Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase. Science. 1990 Sep 14;249(4974):1261–1265. doi: 10.1126/science.2399463. [DOI] [PubMed] [Google Scholar]
  31. Peck L. J., Wang J. C. Energetics of B-to-Z transition in DNA. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6206–6210. doi: 10.1073/pnas.80.20.6206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pourcher T., Sarkar H. K., Bassilana M., Kaback H. R., Leblanc G. Histidine-94 is the only important histidine residue in the melibiose permease of Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jan;87(1):468–472. doi: 10.1073/pnas.87.1.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pruss G. J. DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling. J Mol Biol. 1985 Sep 5;185(1):51–63. doi: 10.1016/0022-2836(85)90182-2. [DOI] [PubMed] [Google Scholar]
  34. Pruss G. J., Drlica K. Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8952–8956. doi: 10.1073/pnas.83.23.8952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Randall L. L., Hardy S. J., Thom J. R. Export of protein: a biochemical view. Annu Rev Microbiol. 1987;41:507–541. doi: 10.1146/annurev.mi.41.100187.002451. [DOI] [PubMed] [Google Scholar]
  37. Randall L. L. Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation. Cell. 1983 May;33(1):231–240. doi: 10.1016/0092-8674(83)90352-5. [DOI] [PubMed] [Google Scholar]
  38. Reaban M. E., Griffin J. A. Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region. Nature. 1990 Nov 22;348(6299):342–344. doi: 10.1038/348342a0. [DOI] [PubMed] [Google Scholar]
  39. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Saier M. H., Jr, Werner P. K., Müller M. Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with the eucaryotic process. Microbiol Rev. 1989 Sep;53(3):333–366. doi: 10.1128/mr.53.3.333-366.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
  42. Schwarz K., Hansen-Hagge T., Bartram C. Improved yields of long PCR products using gene 32 protein. Nucleic Acids Res. 1990 Feb 25;18(4):1079–1079. doi: 10.1093/nar/18.4.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stochaj U., Ehring R. The N-terminal region of Escherichia coli lactose permease mediates membrane contact of the nascent polypeptide chain. Eur J Biochem. 1987 Mar 16;163(3):653–658. doi: 10.1111/j.1432-1033.1987.tb10914.x. [DOI] [PubMed] [Google Scholar]
  45. Stochaj U., Fritz H. J., Heibach C., Markgraf M., von Schaewen A., Sonnewald U., Ehring R. Truncated forms of Escherichia coli lactose permease: models for study of biosynthesis and membrane insertion. J Bacteriol. 1988 Jun;170(6):2639–2645. doi: 10.1128/jb.170.6.2639-2645.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang J. C. DNA topoisomerases: nature's solution to the topological ramifications of the double-helix structure of DNA. Harvey Lect. 1985 1986;81:93–110. [PubMed] [Google Scholar]
  48. Wang J. C. Interaction between DNA and an Escherichia coli protein omega. J Mol Biol. 1971 Feb 14;55(3):523–533. doi: 10.1016/0022-2836(71)90334-2. [DOI] [PubMed] [Google Scholar]
  49. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  50. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]
  51. Yamaguchi A., Akasaka T., Ono N., Someya Y., Nakatani M., Sawai T. Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices. J Biol Chem. 1992 Apr 15;267(11):7490–7498. [PubMed] [Google Scholar]
  52. Yazyu H., Shiota-Niiya S., Shimamoto T., Kanazawa H., Futai M., Tsuchiya T. Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli. J Biol Chem. 1984 Apr 10;259(7):4320–4326. [PubMed] [Google Scholar]
  53. Zabarovsky E. R., Winberg G. High efficiency electroporation of ligated DNA into bacteria. Nucleic Acids Res. 1990 Oct 11;18(19):5912–5912. doi: 10.1093/nar/18.19.5912. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES