1887

Abstract

SUMMARY: mutants of K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml. APase is a phosphate repressible periplasmic enzyme encoded by the gene . With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic strains containing either a constitutive regulatory mutation, or a hybrid plasmid carrying the structural gene and regulatory genes, or a transducing ϕ 80 phage. In the presence of the mutation, F strains lysogenized with ϕ 80 phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml) than haploid strain 2336 ( ) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.

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1986-01-01
2024-12-02
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References

  1. Amemura M., Shinagawa H., Makino K., Otsuji N., Nakata A. 1982; Cloning and complementation tests with alkaline phosphatase regulatory gene (phoS and phoT) of Escherichia coli. Journal of Bacteriology 152:692–701.
    [Google Scholar]
  2. Argast M., Boos W. 1980; Co-regulation in Escherichia coli of a novel transport system for snglycerol-3-phosphate and outer membrane protein Ic (e, E) with alkaline phosphatase and phosphatebinding protein. Journal of Bacteriology 143:142–150.
    [Google Scholar]
  3. Atlan D., Portailer R. 1984; Optimized extracellular production of alkaline phosphatase by lky mutants of Escherichia coli K 12. Applied Microbiology and Biotechnology 19:5–12.
    [Google Scholar]
  4. Berg P. 1981; Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase. Journal of Bacteriology 146:660–667
    [Google Scholar]
  5. Brickman E., Oliver D., Garwin J., Kumamoto C., Beckwith J. 1984; The use of extragenic suppressors to define genes involved in protein export in Escherichia coli. Molecular and General Genetics 196:24–27
    [Google Scholar]
  6. Fognini-Lefebvre N., Portalier R. 1984; Isolation and preliminary characterization of βlactamase periplasmic-leaky mutants of E. coli K-12. FEMS Microbiology Letters 21:323–328
    [Google Scholar]
  7. Glenn A. R. 1976; Production of extracellular proteins by bacteria. Annual Review of Microbiology 30:41–62
    [Google Scholar]
  8. Inouye H., michaelis S., Wright A., Beckwith J. 1981; Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro. Journal of Bacteriology 146:668–675
    [Google Scholar]
  9. Inouye H., Barnes W., Beckwith J. 1982; Signal sequence of alkaline phosphatase of Escherichia coli. Journal of Bacteriology 149:434–439
    [Google Scholar]
  10. Laemmli U. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  11. Lazzaroni J. C., Portalier R. 1979; Isolation and preliminary characterization of periplasmicleaky mutants of Escherichia coli K 12. FEMS Microbiology Letters 5:411–416
    [Google Scholar]
  12. Lazzaroni J. C., Portalier R. 1981; Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K 12. Journal of Bacteriolgy 145:1351–1358
    [Google Scholar]
  13. Lazzaroni J. C., Portalier R. 1982; Production of extracellular alkaline phosphatase by Escherichia coli K 12 periplasmic-leaky mutants carrying phoA + plasmids. European Journal of Applied Microbiology arid Biotechnology 16:146–150
    [Google Scholar]
  14. Ludtke D., Bernstein J., Hamilton C., Torriani A. 1984; Identification of the phoM gene product and its regulation in Escherichia coli K 12. Journal of Bacteriology 159:19–25
    [Google Scholar]
  15. Lugtenberg B. 1981; Composition and function of the outer membrane of Escherichia coli. Trends in Biochemical Sciences 6:262–266
    [Google Scholar]
  16. Miller J. 1972; Experiments in Molecular Genetics. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
    [Google Scholar]
  17. Nakata A., Amemura M., Shinagawa H. 1984; Regulation of the phosphate regulon in Escherichia coli K 12: Regulation of the negative regulatory gene phoU and identification of the gene product. Journal of Bacteriology 159:979–985
    [Google Scholar]
  18. Shinagawa H., Makino K., Nakata A. 1983; Regulation of the pho regulon in Escherichia coli K 12. Genetic and physiological regulation of the positive regulatory gene (phoB) expression. Journal of Molecular Biology 168:477–488
    [Google Scholar]
  19. Silhavy T., Benson S., Emr S. 1983; Mechanisms of protein localization. Microbiological Reviews 47:313–344
    [Google Scholar]
  20. Tommassen J., Lugtenberg B. 1982; pho regulon of Escherichia coli K12: a minireview. Annales de Microbiologie 133A:243–249
    [Google Scholar]
  21. Torriani A. M. 1967; Alkaline phosphatase from Escherichia coli. In Procedures in Nucleic Acid Research, Edited by G. L. Cantoni & D. R. Davies, New York: Harper and Row. 224–235
    [Google Scholar]
  22. Wanner B., Sarthy A., Beckwith J. 1979; Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins. Journal of Bacteriology 140:229–239
    [Google Scholar]
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