Molecular and General Genetics MGG

, Volume 204, Issue 1, pp 166–173 | Cite as

Studies on lipase directed export of Escherichia coli β-lactamase in Staphylococcus carnosus

  • Wolfgang Liebl
  • Friedrich Götz


The lipase (lip) gene of Staphylococcus hyicus was used to study the expression of the Escherichia coli β-lactamase (bla) gene in S. carnosus. The bla gene, devoid of its promotor and most of the signal sequence, was fused to the lip structural gene at various positions. A set of 11 secretion vectors (pLLβ1 to pLLβ11) was isolated and analysed. All secretion vectors caused β-lactamase production and activity in S. carnosus. However, the amount of hybrid proteins secreted was influenced by the length of the NH2-terminal lipase portion. An increased concentration, comparable to that of the native lipase, of secreted lipase/β-lactamase hybrid proteins was only found when the lipase portion of the construct comprised more than 101 amino acids of the NH2-terminal region of the lipase preprotein; the proposed lipase signal peptide is 36 amino acids long. If the hybrid proteins constructed contained 101 or less amino acids of the NH2-terminal lipase preprotein, only low amounts of secreted hybrid proteins were detectable and a significant portion of the hybrid proteins and β-lactamase activity was found in the cellular fraction. The results indicate that the lipase possesses adjacent to the signal peptide a peptide domain that is essential for the secretion of the lipase/β-lactamase hybrid proteins.

Key words

Gene expression Lipase gene fusion Protein export Secretion vector Staphylococcus carnosus 




bla gene

beta lactamase coding gene of Escherichia coli

lip gene

lipase-coding gene of Staphylococcus hyicus




PA gel electrophoresis


sodium dodecyl sulphate


indicates plasmid-carrier state


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Blake MS, Johnston KH, Russel-Jones GJ, Gotschlich EC (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 136:175–179Google Scholar
  2. Boyko WL, Ganschow RE (1982) Rapid identification of Escherichia coli transformed by pBR322 carrying inserts at the PstI site. Anal Biochem 122:85–88Google Scholar
  3. Chang NC, Nielsen JBK, Izu K, Blobel G, Lampen JO (1982) Identification of the signal peptidase cleavage site in Bacillus licheniformis prepenicillinase. J Biol Chem 257:4340–4344Google Scholar
  4. Götz F, Zabielski J, Philipson L, Lindberg M (1983a) DNA homology between the arsenate resistance plasmid pSX267 from Staphylococcus xylosus and the penicillinase plasmid pI258 from Staphylococcus aureus. Plasmid 9:126–137Google Scholar
  5. Götz F, Kreutz B, Schleifer KH (1983b) Protoplast transformation of Staphylococcus carnosus by plasmid DNA. Mol Gen Genet 189:340–342Google Scholar
  6. Götz F, Popp F, Korn E, Schleifer KH (1985) Complete nucleotide sequence of the lipase gene from Staphylococcus hyicus cloned in Staphylococcus carnosus. Nucl Acids Res 13:5895–5906Google Scholar
  7. Hantke K, Braun V (1973) Covalent binding of lipid to protein: Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur J Biochem 34:284–296Google Scholar
  8. Kadonaga JT, Gautier AE, Straus DR, Charles AD, Edge MD, Knowles JR (1984) The role of the β-lactamase signal sequence in the secretion of proteins by Escherichia coli. J Biol Chem 259:2149–2154Google Scholar
  9. Keller G, Schleifer KH, Götz F (1983) Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus. Plasmid 10:270–278Google Scholar
  10. Keller G, Schleifer KH, Götz F (1984) Cloning of the ribokinase gene of Staphylococcus hyicus subsp. hyicus in Staphylococcus carnosus. Arch Microbiol 140:218–224Google Scholar
  11. Koshland D, Botstein D (1982) Evidence for posttranslational translocation of β-lactamase across the bacterial inner membrane. Cell 30:893–902Google Scholar
  12. Koshland D, Sauer RT, Botstein D (1982) Diverse effects of mutations in the signal sequence on the secretion of β-lactamase in Salmonella typhimurium. Cell 30:903–914Google Scholar
  13. Kreutz B, Götz F (1984) Construction of Staphylococcus plasmid vector pCA43 conferring resistance to chloramphenicol, arsenate, arsenite and antimony. Gene 31:301–304Google Scholar
  14. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  15. Liepe H-U (1982) Starter cultures and dry sausage. Forum Mikrobiol 5:10–15Google Scholar
  16. Maxam AM, Gilbert W (1980) Sequencing end labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560Google Scholar
  17. Neugebauer K, Sprengel R, Schaller H (1981) Penicillinase from Bacillus licheniformis: Nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Grampositive bacterium. Nucleic Acids Res 9:2577–2588Google Scholar
  18. Nielsen JBK, Lampen JO (1982a) Membrane-bound penicillinases in Gram-positive bacteria. J Biol Chem 257:4490–4495Google Scholar
  19. Nielsen JBK, Lampen JO (1982b) Glyceride-cysteine lipoproteins and secretion by Gram-positive bacteria. J Bacteriol 152:315–322Google Scholar
  20. O'Callaghan CH, Morris A, Kirby SM, Shingler AH (1972) Novel method for detection of β-lactamase by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1:283–288Google Scholar
  21. Schleifer KH, Fischer U (1982) Description of a new species of the genus Staphylococcus: Staphylococcus carnosus. Int J Syst Bacteriol 32:153–156Google Scholar
  22. Sutcliffe JG (1978) Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci USA 75:3737–3741Google Scholar
  23. Sutcliffe JG (1979) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90Google Scholar
  24. Thudt K, Schleifer KH, Götz F (1985) Cloning and expression of alpha amylase gene from Bacillus stearothermophilus in Staphylococcus carnosus and other staphylococcal species. Gene 37:163–169Google Scholar
  25. Towbin H, Stachelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • Wolfgang Liebl
    • 1
  • Friedrich Götz
    • 1
  1. 1.Lehrstuhl für MikrobiologieTechnische Universität MünchenMünchen 2Federal Republic of Germany

Personalised recommendations