Advertisement

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
Article

Summary

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 

Abbreviations

Cm

chloramphenicol

bla gene

beta lactamase coding gene of Escherichia coli

lip gene

lipase-coding gene of Staphylococcus hyicus

PA

polyacrylamide

PAGE

PA gel electrophoresis

SDS

sodium dodecyl sulphate

[]

indicates plasmid-carrier state

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  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