Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1983 May;154(2):946–954. doi: 10.1128/jb.154.2.946-954.1983

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

R Kumar, A Ghosh, B K Ghosh
PMCID: PMC217549  PMID: 6188749

Abstract

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.

Full text

PDF
946

Images in this article

950Image
on p.950
952Image
on p.952

Selected References

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

  1. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carne T., Scheele G. Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins. J Biol Chem. 1982 Apr 25;257(8):4133–4140. [PubMed] [Google Scholar]
  3. Cerdá-Olmedo E., Hanawalt P. C. The replication of the Escherichia coli chromosome studied by sequential nitrosoguanidine mutagenesis. Cold Spring Harb Symp Quant Biol. 1968;33:599–607. doi: 10.1101/sqb.1968.033.01.066. [DOI] [PubMed] [Google Scholar]
  4. Chang C. N., Nielsen J. B., Izui K., Blobel G., Lampen J. O. Identification of the signal peptidase cleavage site in Bacillus licheniformis prepenicillinase. J Biol Chem. 1982 Apr 25;257(8):4340–4344. [PubMed] [Google Scholar]
  5. Friedman J., Huberman E. Postreplication repair and the susceptibility of Chinese hamster cells to cytotoxic and mutagenic effects of alkylating agents. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6072–6076. doi: 10.1073/pnas.77.10.6072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ghosh A., Ghosh B. K. Immunoelectron microscopic localization of penicillinase in Bacillus licheniformis. J Bacteriol. 1979 Mar;137(3):1374–1385. doi: 10.1128/jb.137.3.1374-1385.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ghosh B. K., Sargent M. G., Lampen J. O. Morphological phenomena associated with penicillinase induction and secretion in Bacillus licheniformis. J Bacteriol. 1968 Oct;96(4):1314–1328. doi: 10.1128/jb.96.4.1314-1328.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ghosh B. K., Wouters J. T., Lampen J. O. Distribution of the sites of alkaline phosphatase(s) activity in vegetative cells of Bacillus subtilis. J Bacteriol. 1971 Nov;108(2):928–937. doi: 10.1128/jb.108.2.928-937.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ghosh R., Ghosh A., Ghosh B. K. Properties of the membrane-bound alkaline phosphatase from glucose- and lactate-grown cells of Bacillus subtilis SB 15. J Biol Chem. 1977 Oct 10;252(19):6813–6822. [PubMed] [Google Scholar]
  10. Guerola N., Ingraham J. L., Cerdá-Olmedo E. Induction of closely linked multiple mutations by nitrosoguanidine. Nat New Biol. 1971 Mar 24;230(12):122–125. doi: 10.1038/newbio230122a0. [DOI] [PubMed] [Google Scholar]
  11. Henner D. J., Hoch J. A. The Bacillus subtilis chromosome. Microbiol Rev. 1980 Mar;44(1):57–82. doi: 10.1128/mr.44.1.57-82.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hydrean C., Ghosh A., Nallin M., Ghosh B. K. Interrelationship of carbohydrate metabolism and alkaline phosphatase synthesis in Bacillus licheniformis 749/c. J Biol Chem. 1977 Oct 10;252(19):6806–6812. [PubMed] [Google Scholar]
  13. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  14. Koshland D., Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. doi: 10.1016/0092-8674(80)90321-9. [DOI] [PubMed] [Google Scholar]
  15. Koyama A. H., Wada C., Nagata T., Yura T. Indirect selection for plasmid mutants: isolation of ColVBtrp mutants defective in self-maintenance in Escherichia coli. J Bacteriol. 1975 Apr;122(1):73–79. doi: 10.1128/jb.122.1.73-79.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Le Hégarat J. C., Anagnostopoulos C. Purification, subunit structure and properties of two repressible phosphohydrolases of Bacillus subtilis. Eur J Biochem. 1973 Nov 15;39(2):525–539. doi: 10.1111/j.1432-1033.1973.tb03151.x. [DOI] [PubMed] [Google Scholar]
  19. Meyer D. I., Dobberstein B. Identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum. J Cell Biol. 1980 Nov;87(2 Pt 1):503–508. doi: 10.1083/jcb.87.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miki T., Minami Z., Ikeda Y. The genetics of alkaline phosphatase formation in Bacillus subtilis. Genetics. 1965 Nov;52(5):1093–1100. doi: 10.1093/genetics/52.5.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mostov K. E., Kraehenbuhl J. P., Blobel G. Receptor-mediated transcellular transport of immunoglobulin: synthesis of secretory component as multiple and larger transmembrane forms. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7257–7261. doi: 10.1073/pnas.77.12.7257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oliver D. B., Beckwith J. Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli. J Bacteriol. 1982 May;150(2):686–691. doi: 10.1128/jb.150.2.686-691.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Oliver D. B., Beckwith J. Regulation of a membrane component required for protein secretion in Escherichia coli. Cell. 1982 Aug;30(1):311–319. doi: 10.1016/0092-8674(82)90037-x. [DOI] [PubMed] [Google Scholar]
  24. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  25. Piggot P. J., Taylor S. Y. New types of mutation affecting formation of alkaline phosphatase by Bacillus subtilis in sporulation conditions. J Gen Microbiol. 1977 Sep;102(1):69–80. doi: 10.1099/00221287-102-1-69. [DOI] [PubMed] [Google Scholar]
  26. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sargent M. G., Ghosh B. K., Lampen J. O. Localization of cell-bound penicillinase in Bacillus licheniformis. J Bacteriol. 1968 Oct;96(4):1329–1338. doi: 10.1128/jb.96.4.1329-1338.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sarthy A., Fowler A., Zabin I., Beckwith J. Use of gene fusions to determine a partial signal sequence of alkaline phosphatase. J Bacteriol. 1979 Sep;139(3):932–939. doi: 10.1128/jb.139.3.932-939.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sarthy A., Michaelis S., Beckwith J. Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA. J Bacteriol. 1981 Jan;145(1):288–292. doi: 10.1128/jb.145.1.288-292.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Scheele G. A. Biosynthesis, segregation, and secretion of exportable proteins by the exocrine pancreas. Am J Physiol. 1980 Jun;238(6):G467–G477. doi: 10.1152/ajpgi.1980.238.6.G467. [DOI] [PubMed] [Google Scholar]
  31. Spencer D. B., Hansa J. G., Stuckmann K. V., Hulett F. M. Membrane-associated alkaline phosphatase from Bacillus licheniformis that requires detergent for solubilization: lactoperoxidase 125I localization and molecular weight determination. J Bacteriol. 1982 May;150(2):826–834. doi: 10.1128/jb.150.2.826-834.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vanderkooi G. Organisation of proteins in membranes with special reference to the cytochrome oxidase system. Biochim Biophys Acta. 1974 Dec 16;344(3-4):307–344. doi: 10.1016/0304-4157(74)90011-2. [DOI] [PubMed] [Google Scholar]
  33. Walter P., Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. doi: 10.1073/pnas.77.12.7112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):551–556. doi: 10.1083/jcb.91.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yamane K., Maruo B. Alkaline phosphatase possessing alkaline phosphodiesterase activity and other phosphodiesterases in Bacillus subtilis. J Bacteriol. 1978 Apr;134(1):108–114. doi: 10.1128/jb.134.1.108-114.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES