Advertisement

Molecular and General Genetics MGG

, Volume 230, Issue 1–2, pp 230–240 | Cite as

Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli

  • Jürgen Robert Klein
  • Bernhard Henrich
  • Roland Plapp
Article

Summary

The chromosomal DNA insert in plasmid pJK131, which complements the phenotypic defects associated with a mutation in the envC gene of Escherichia coli strain PM61, was sequenced. The analysis of the nucleotide sequence revealed two open reading frames (ORFs) coding for the proteins EnvC (41281 daltons) and EnvD (104415 daltons). The envC gene product is synthesized as a pre-protein and, after cleavage of a signal peptide, the mature protein is incorporated into the cytoplasmic membrane. The detection of a common transcript for both ORFs indicated the existence of an envCD operon. Deletion analysis and the generation of frameshifts demonstrated that simultaneous expression of both genes is required to complement the defects in strain PM61. Overproduction of EnvC protein appears to be lethal to Escherichia coli. The envD gene, however, could be cloned and expressed at high levels under control of the tac promotor without deleterious effects on the host.

Key words

envCD operon Nucleotide sequence EnvC protein Membrane localization 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Argos P, Rao JKM, Hargrave PA (1982) Structural prediction of membrane-bound proteins. Eur J Biochem 128:565–575Google Scholar
  2. Bachman BJ (1990) Linkage map of Escherichia coli K-12, Edition 8. Microbiol Rev 54:130–197Google Scholar
  3. Beck E, Bremer E (1980) Nucleotide sequence of the gene ompA coding the outer membrane protein II of Escherichia coli K-12. Nucleic Acids Res 8:3011–3024Google Scholar
  4. Bennetzen JL, Hall BD (1982) Codon selection in yeast. J Biol Chem 257:3026–3031Google Scholar
  5. Birkenbihl RP, Vielmetter W (1989) Complete maps of IS1, IS2, IS3, IS4, IS5, IS30 and IS150 locations in Escherichia coli K12. Mol Gen Genet 220:147–153Google Scholar
  6. de Boer PAJ, Cook WR, Rothfield LI (1990) Bacterial cell division. Annu Rev Genet 24:249–274Google Scholar
  7. Broome-Smith JK, Ioannidis I, Edelman A, Spratt BG (1988) Nucleotide sequence of the penicillin-binding protein 5 and 6 genes of Escherichia coli. Nucleic Acids Res 16:1617Google Scholar
  8. Donachie WD, Robinson AC (1987) Cell division: Parameter values and the process. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. American Society for Microbiology, Washington, DC, pp 1578–1593Google Scholar
  9. Duffand GD, Lehnhardt SK, March PE, Inouye M (1985) Structure and function of the signal peptide. Curr Top Membranes Transport 24:65–104Google Scholar
  10. Filip C, Fletcher G, Wulf JL, Earhart CF (1973) Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115:717–722Google Scholar
  11. Gribskov M, Dvereux J, Burgess RR (1984) The codon preference plot: Graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res 12:539–549Google Scholar
  12. Gustafsson P, Nordström K, Normark S (1973) Outer penetration barrier of Escherichia coli K-12: Kinetics of the uptake of gentian violet by wild type and envelope mutants. J Bacteriol 116:893–900Google Scholar
  13. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580Google Scholar
  14. Henikoff D (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359Google Scholar
  15. Henrich B, Monnerjahn U, Plapp R (1990) Peptidase D gene (pepD) of Escherichia coli K-12: Nucleotide sequence, transcript mapping, and comparison with other peptidase genes. J Bacteriol 172:4641–4651Google Scholar
  16. Ito K, Sato T, Yura T (1977) Synthesis of the membrane proteins in E. coli. Cell 11:551–559Google Scholar
  17. Karibian D, Pellon G, Starka J (1981) Autolysis of a division mutant of Escherichia coli. J Gen Microbiol 126:55–61Google Scholar
  18. Karibian D, Michel GPF, Starka J (1985) The envC phenotype and its expression in various Escherichia coli K12 strains. FEMS Microbiol Lett 27:319–324Google Scholar
  19. Klein JR, Henrich B, Plapp R (1990) Molecular cloning of the envC gene of Escherichia coli. Curr Microbiol 21:341–347Google Scholar
  20. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132Google Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  22. Levy SB, McMurry L (1974) Detection of an inducible membrane protein associated with R factor-mediated tetracycline resistance. Biochem Biophys Res Commun 56:1060–1068Google Scholar
  23. Luktenhaus J (1988) Genetic analysis of bacterial cell division. Microbiol Sci 5:88–91Google Scholar
  24. Luktenhaus J (1990) Regulation of cell division in E. coli. Trends Genet 6:22–25Google Scholar
  25. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  26. Meselson M, Yuan R (1968) DNA restriction enzyme from Escherichia coli. Nature 217:1110–1114Google Scholar
  27. Messing J, Vieira J (1982) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276Google Scholar
  28. Michel G, Starka J (1979) Phospholipase A activity with integrated phospholipid vesicles in intact cells of an envelope mutant of Escherichia coli. FEBS Lett 108:261–265Google Scholar
  29. Michel G, Di Savino D, Starka J (1977) Phospholipid composition and phenotypic correction of an envC mutant of Escherichia coli. J Bacteriol 129:145–150Google Scholar
  30. Neu HC, Happel LA (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 240:3685–3692Google Scholar
  31. Oliver BD (1987) Periplasm and protein secretion. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium. Cellular and molecular biology. American Society for Microbiology, Washington, DC, pp 56–69Google Scholar
  32. Osborn MJ, Munson R (1974) Separation of inner and outer membranes of Gram-negative bacteria. Methods Enzymol 31:642–653Google Scholar
  33. Pugsley AP (1989) Protein Targeting. Academic Press, London, pp 45–111Google Scholar
  34. Queen C, Korn LJ (1984) A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res 12:581–599Google Scholar
  35. Rodolakis A, Thomas B, Starka J (1973) Morphological mutants of Escherichia coli. Isolation and ultrastructure of a chain-forming envC mutant. J Gen Microbiol 75:409–416Google Scholar
  36. Rodolakis A, Casse F, Starka J (1974) Morphological mutants of Escherichia coli K12. Mapping of the envC mutation. Mol Gen Genet 130:177–181Google Scholar
  37. Rothfield L, Romeo D (1971) Enzyme reactions in biological membranes. In: Rothfield L (ed) Structure and function of biological membranes. Academic Press, New York, pp 251–284Google Scholar
  38. Russel DR, Bennet GN (1982) Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the −35 to −10 spacing. Gene 20:231–243Google Scholar
  39. Sabatini DD, Kreibich G, Morimoto T, Adesnik M (1982) Mechanism for the incorporation of proteins in membranes and organelles. J Cell Biol 92:1–22Google Scholar
  40. Saier MH, Werner PK, Müller M (1990) Insertion of proteins into bacterial membranes: Mechanism, characteristics, and comparisons with the eucaryotic process. Microbiol Rev 53:333–366Google Scholar
  41. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  42. Spratt BG, Boyd A, Stoker N (1980) Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-pbpA-leuS region of the Escherichia coli chromosome. J Bacteriol 143:569–581Google Scholar
  43. Staden R (1984) Measurements of the effects that coding for a protein has on a DNA sequence and their use for finding genes. Nucleic Acids Res 12:551–567Google Scholar
  44. Starkova Z, Thomas P, Starka J (1978) Morphological mutants of Escherichia coli: Nature of the permeability barrier in mon and envC cells. Ann Inst Pasteur Microbiol 129:265–284Google Scholar
  45. Starkova Z, Bonnaveiro N, Starka J (1981) Hydrolysis of phospholipids by phospholipase C in intact cells of wild-type and envelope mutants of Escherichia coli K12. FEBS Lett 130:261–264Google Scholar
  46. Stoker NG, Fairweather NF, Spratt BG (1982) Versatile low-copynumber plasmid vectors for cloning in Escherichia coli. Gene 18:335–341Google Scholar
  47. Wadle D, Henrich B, Plapp R (1986) Effects of mutations in genes fadR, fadE, and envC of Escherichia coli on the action of the lysis gene of bacteriophage ΦX174. Curr Microbiol 14:65–69Google Scholar
  48. van Wezenbeek PMGF, Hulsebos TJM, Schoenmakers JGG (1980) Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: Comparison with phage fd. Gene 11:129–148Google Scholar
  49. Wollman EL, Jacob F (1956) Conjugation and genetic recombination in Escherichia coli K-12. Cold Spring Harbor Symp Quant Biol 21:141–162Google Scholar
  50. Wu HC, Tokunaga M (1986) Biogenesis of lipoproteins in bacteria. Curr Top Microbiol Immunol 125:127–158Google Scholar
  51. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 33:103–119Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Jürgen Robert Klein
    • 1
  • Bernhard Henrich
    • 1
  • Roland Plapp
    • 1
  1. 1.Fachbereich Biologie, Abteilung MikrobiologieUniversität KaiserslauternKaiserslauternGermany

Personalised recommendations