Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Sep;169(9):4135–4140. doi: 10.1128/jb.169.9.4135-4140.1987

Membrane protein binding to the origin region of Bacillus subtilis.

J Laffan, W Firshein
PMCID: PMC213720  PMID: 3114234

Abstract

Binding of membrane proteins extracted from Bacillus subtilis to an 11.6-kilobase region containing the origin of replication was examined by Western blotting (protein blotting) procedures. Two adjacent origin probes in the double-stranded form (spanning a length of 4 kilobases) were found to bind very strongly to a 63-kilodalton (kDa) protein in that they resisted dissociation after a high-concentration salt wash. This region encompasses both a site implicated in initiation in vivo and a gene coding for a DNA gyrase subunit (gyrA). In contrast, flanking origin and nonorigin double-stranded probes were dissociated after washing with a high salt concentration. Another protein of 67 kDa bound less intensely to the putative initiation site but not to the gyrA region. All of the origin and nonorigin probes in the double- or single-stranded form were found to bind nonspecifically to a subset of 10 to 12 proteins of 50 to 60 separated by gel electrophoresis after a low-concentration salt wash. They ranged in size from 14 to over 100 kDa (including 63 kDa). However, in contrast to the double-stranded forms, most of the single-stranded probes resisted dissociation from the protein subset after a high-concentration salt wash.

Full text

PDF
4135

Images in this article

4137Image
on p.4137
4137Image
on p.4137
4137Image
on p.4137
4138Image
on p.4138

Selected References

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

  1. Alberts B. M., Frey L. T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature. 1970 Sep 26;227(5265):1313–1318. doi: 10.1038/2271313a0. [DOI] [PubMed] [Google Scholar]
  2. Benjamin P., Firshein W. Initiation of DNA replication in vitro by a DNA-membrane complex extracted from Bacillus subtilis. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6214–6218. doi: 10.1073/pnas.80.20.6214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benjamin P., Strumph P., Kenny M., Firshein W. DNA synthesis in purified DNA-membrane complexes extracted from a Bacillus subtilis pol A mutant. Nature. 1982 Aug 19;298(5876):769–771. doi: 10.1038/298769a0. [DOI] [PubMed] [Google Scholar]
  4. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Echols H. Multiple DNA-protein interactions governing high-precision DNA transactions. Science. 1986 Sep 5;233(4768):1050–1056. doi: 10.1126/science.2943018. [DOI] [PubMed] [Google Scholar]
  6. Firshein W. The DNA-membrane fraction of Pneumococcus contains a DNA replication complex. J Mol Biol. 1972 Oct 14;70(3):383–397. doi: 10.1016/0022-2836(72)90547-5. [DOI] [PubMed] [Google Scholar]
  7. Fuller R. S., Funnell B. E., Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  8. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jacq A., Kohiyama M., Lother H., Messer W. Recognition sites for a membrane-derived DNA binding protein preparation in the E. coli replication origin. Mol Gen Genet. 1983;191(3):460–465. doi: 10.1007/BF00425763. [DOI] [PubMed] [Google Scholar]
  10. Korn R., Winston S., Tanaka T., Sueoka N. Specific in vitro binding of a plasmid to a membrane fraction of Bacillus subtilis. Proc Natl Acad Sci U S A. 1983 Jan;80(2):574–578. doi: 10.1073/pnas.80.2.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kornacki J. A., Firshein W. Replication of plasmid RK2 in vitro by a DNA-membrane complex: evidence for initiation of replication and its coupling to transcription and translation. J Bacteriol. 1986 Jul;167(1):319–326. doi: 10.1128/jb.167.1.319-326.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kusano T., Steinmetz D., Hendrickson W. G., Murchie J., King M., Benson A., Schaechter M. Direct evidence for specific binding of the replicative origin of the Escherichia coli chromosome to the membrane. J Bacteriol. 1984 Apr;158(1):313–316. doi: 10.1128/jb.158.1.313-316.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Laffan J., Firshein W. DNA replication by a DNA-membrane complex extracted from Bacillus subtilis: site of initiation in vitro and initiation potential of subcomplexes. J Bacteriol. 1987 Jun;169(6):2819–2827. doi: 10.1128/jb.169.6.2819-2827.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lampe M. F., Bott K. F. Genetic and physical organization of the cloned gyrA and gyrB genes of Bacillus subtilis. J Bacteriol. 1985 Apr;162(1):78–84. doi: 10.1128/jb.162.1.78-84.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leibowitz P. J., Schaechter M. The attachment of the bacterial chromosome to the cell membrane. Int Rev Cytol. 1975;41:1–28. doi: 10.1016/s0074-7696(08)60964-x. [DOI] [PubMed] [Google Scholar]
  17. Marty-Mazars D., Horiuchi S., Tai P. C., Davis B. D. Proteins of ribosome-bearing and free-membrane domains in Bacillus subtilis. J Bacteriol. 1983 Jun;154(3):1381–1388. doi: 10.1128/jb.154.3.1381-1388.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Moriya S., Ogasawara N., Yoshikawa H. Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region. Nucleic Acids Res. 1985 Apr 11;13(7):2251–2265. doi: 10.1093/nar/13.7.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ogasawara N., Mizumoto S., Yoshikawa H. Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome. Gene. 1984 Oct;30(1-3):173–182. doi: 10.1016/0378-1119(84)90118-5. [DOI] [PubMed] [Google Scholar]
  20. Seiki M., Ogasawara N., Yoshikawa H. Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. I. Isolation and characterization of plasmids containing the origin region. Mol Gen Genet. 1981;183(2):220–226. doi: 10.1007/BF00270621. [DOI] [PubMed] [Google Scholar]
  21. Sigal N., Delius H., Kornberg T., Gefter M. L., Alberts B. A DNA-unwinding protein isolated from Escherichia coli: its interaction with DNA and with DNA polymerases. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3537–3541. doi: 10.1073/pnas.69.12.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stewart G. C., Bott K. F. DNA sequence of the tandem ribosomal RNA promoter for B. subtilis operon rrnB. Nucleic Acids Res. 1983 Sep 24;11(18):6289–6300. doi: 10.1093/nar/11.18.6289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Séror-Laurent S. J., Henckes G. An RNA-DNA copolymer whose synthesis is correlated with the transcriptional requirement for chromosomal initiation in Bacillus subtilis contains ribosomal RNA sequences. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3586–3590. doi: 10.1073/pnas.82.11.3586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tremblay G. Y., Daniels M. J., Schaechter M. Isolation of a cell membrane-DNA-nascent RNA complex from bacteria. J Mol Biol. 1969 Feb 28;40(1):65–76. doi: 10.1016/0022-2836(69)90296-4. [DOI] [PubMed] [Google Scholar]
  25. Weber K., Kuter D. J. Reversible denaturation of enzymes by sodium dodecyl sulfate. J Biol Chem. 1971 Jul 25;246(14):4504–4509. [PubMed] [Google Scholar]
  26. Weiss A. S., Wake R. G. Restriction map of DNA spanning the replication terminus of the Bacillus subtilis chromosome. J Mol Biol. 1983 Dec 5;171(2):119–137. doi: 10.1016/s0022-2836(83)80349-0. [DOI] [PubMed] [Google Scholar]
  27. Witholt B., Heerikhuizen H. V., De Leij L. How does lysozyme penetrate through the bacterial outer membrane? Biochim Biophys Acta. 1976 Sep 7;443(3):534–544. doi: 10.1016/0005-2736(76)90471-5. [DOI] [PubMed] [Google Scholar]
  28. Zylicz M., Yamamoto T., McKittrick N., Sell S., Georgopoulos C. Purification and properties of the dnaJ replication protein of Escherichia coli. J Biol Chem. 1985 Jun 25;260(12):7591–7598. [PubMed] [Google Scholar]

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

RESOURCES