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. 1989 Dec;171(12):6747–6752. doi: 10.1128/jb.171.12.6747-6752.1989

In vitro reconstitution of a hexagonal array with a surface layer protein synthesized by Bacillus subtilis harboring the surface layer protein gene from Bacillus brevis 47.

A Tsuboi 1, R Uchihi 1, H Engelhardt 1, H Hattori 1, S Shimizu 1, N Tsukagoshi 1, S Udaka 1
PMCID: PMC210572  PMID: 2512285

Abstract

Bacillus brevis 47 contains two surface layer proteins, termed the outer wall protein and the middle wall protein (MWP), which form a hexagonal array in the cell wall. Introduction of the MWP structural gene into Bacillus subtilis by using a low-copy-number plasmid led to the synthesis of an immunoreactive polypeptide with a molecular mass almost the same as that of the MWP synthesized by B. brevis 47. Biochemical analysis indicated that most of the MWP synthesized by B. subtilis was localized in the cytoplasmic fraction. This was further confirmed by using immunogold electron microscopy. The amino-terminal amino acid sequence of the MWP purified from the cytoplasm of B. subtilis indicated that the MWP was precursor with a signal peptide of 23 amino acid residues to the amino terminus of the mature protein. The precursor of the MWP possessed the ability to reassemble in vitro on the B. brevis 47 peptidoglycan layer, resulting in the formation of almost the same hexagonal arrays as with the mature MWP purified from B. brevis 47, judging from images averaged at a resolution of about 2.5 nm. Furthermore, a center-to-center distance of the hexagonal lattice on the envelope reconstituted by using the precursor MWP was calibrated as 18.3 nm, which was almost identical to the value of 17.8 nm obtained with the mature protein.

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Selected References

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

  1. Adachi T., Yamagata H., Tsukagoshi N., Udaka S. Multiple and tandemly arranged promoters of the cell wall protein gene operon in Bacillus brevis 47. J Bacteriol. 1989 Feb;171(2):1010–1016. doi: 10.1128/jb.171.2.1010-1016.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baumeister W., Wildhaber I., Engelhardt H. Bacterial surface proteins. Some structural, functional and evolutionary aspects. Biophys Chem. 1988 Feb;29(1-2):39–49. doi: 10.1016/0301-4622(88)87023-6. [DOI] [PubMed] [Google Scholar]
  3. Beveridge T. J. Ultrastructure, chemistry, and function of the bacterial wall. Int Rev Cytol. 1981;72:229–317. doi: 10.1016/s0074-7696(08)61198-5. [DOI] [PubMed] [Google Scholar]
  4. Kessel M., Radermacher M., Frank J. The structure of the stalk surface layer of a brine pond microorganism: correlation averaging applied to a double layered lattice structure. J Microsc. 1985 Jul;139(Pt 1):63–74. doi: 10.1111/j.1365-2818.1985.tb04662.x. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Lechner J., Sumper M. The primary structure of a procaryotic glycoprotein. Cloning and sequencing of the cell surface glycoprotein gene of halobacteria. J Biol Chem. 1987 Jul 15;262(20):9724–9729. [PubMed] [Google Scholar]
  8. McCurdy D. W., Pratt L. H. Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability. J Cell Biol. 1986 Dec;103(6 Pt 1):2541–2550. doi: 10.1083/jcb.103.6.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Peters J., Peters M., Lottspeich F., Schäfer W., Baumeister W. Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies. J Bacteriol. 1987 Nov;169(11):5216–5223. doi: 10.1128/jb.169.11.5216-5223.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. STEMPAK J. G., WARD R. T. AN IMPROVED STAINING METHOD FOR ELECTRON MICROSCOPY. J Cell Biol. 1964 Sep;22:697–701. doi: 10.1083/jcb.22.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Saxton W. O., Baumeister W. The correlation averaging of a regularly arranged bacterial cell envelope protein. J Microsc. 1982 Aug;127(Pt 2):127–138. doi: 10.1111/j.1365-2818.1982.tb00405.x. [DOI] [PubMed] [Google Scholar]
  12. Schwarz U., Asmus A., Frank H. Autolytic enzymes and cell division of Escherichia coli. J Mol Biol. 1969 May 14;41(3):419–429. doi: 10.1016/0022-2836(69)90285-x. [DOI] [PubMed] [Google Scholar]
  13. Sleytr U. B., Messner P. Crystalline surface layers on bacteria. Annu Rev Microbiol. 1983;37:311–339. doi: 10.1146/annurev.mi.37.100183.001523. [DOI] [PubMed] [Google Scholar]
  14. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tsuboi A., Tsukagoshi N., Udaka S. Reassembly in vitro of hexagonal surface arrays in a protein-producing bacterium, Bacillus brevis 47. J Bacteriol. 1982 Sep;151(3):1485–1497. doi: 10.1128/jb.151.3.1485-1497.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tsuboi A., Uchihi R., Adachi T., Sasaki T., Hayakawa S., Yamagata H., Tsukagoshi N., Udaka S. Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene. J Bacteriol. 1988 Feb;170(2):935–945. doi: 10.1128/jb.170.2.935-945.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tsuboi A., Uchihi R., Tabata R., Takahashi Y., Hashiba H., Sasaki T., Yamagata H., Tsukagoshi N., Udaka S. Characterization of the genes coding for two major cell wall proteins from protein-producing Bacillus brevis 47: complete nucleotide sequence of the outer wall protein gene. J Bacteriol. 1986 Oct;168(1):365–373. doi: 10.1128/jb.168.1.365-373.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Yamada H., Tsukagoshi N., Udaka S. Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47. J Bacteriol. 1981 Oct;148(1):322–332. doi: 10.1128/jb.148.1.322-332.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yamagata H., Adachi T., Tsuboi A., Takao M., Sasaki T., Tsukagoshi N., Udaka S. Cloning and characterization of the 5' region of the cell wall protein gene operon in Bacillus brevis 47. J Bacteriol. 1987 Mar;169(3):1239–1245. doi: 10.1128/jb.169.3.1239-1245.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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