Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Mar;171(3):1409–1416. doi: 10.1128/jb.171.3.1409-1416.1989

Purification and characterization of a low-molecular-weight membrane protein with affinity for the Escherichia coli origin of replication.

A Jacq 1, R Kern 1, A Tsugita 1, M Kohiyama 1
PMCID: PMC209760  PMID: 2646282

Abstract

A purification procedure was devised for a low-molecular-mass (about 10-kilodalton) membrane protein from Escherichia coli that was shown to bind specifically to the chromosomal replication origin region (oriC). Nitrocellulose membrane retention assays showed the binding site to be adjacent to the right boundary of the oriC minimal sequence. We determined the amino acid sequence of the N-terminal and C-terminal regions as well as the global amino acid composition of this membrane protein. Specific antibodies against the protein were produced and used to confirm the cell membrane location of the protein. These results demonstrate that this is a new membrane protein, different from the previously described B' protein, with specific binding activity for the oriC region. We propose that this protein be called membrane oriC-binding protein 2 (MOB2 protein).

Full text

PDF
1414

Images in this article

1411Image
on p.1411
1412Image
on p.1412
1413Image
on p.1413

Selected References

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

  1. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Georgopoulos C. P. A new bacterial gene (groPC) which affects lambda DNA replication. Mol Gen Genet. 1977 Feb 28;151(1):35–39. doi: 10.1007/BF00446910. [DOI] [PubMed] [Google Scholar]
  5. Georgopoulos C., Tilly K., Drahos D., Hendrix R. The B66.0 protein of Escherichia coli is the product of the dnaK+ gene. J Bacteriol. 1982 Mar;149(3):1175–1177. doi: 10.1128/jb.149.3.1175-1177.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. HIRS C. H. The oxidation of ribonuclease with performic acid. J Biol Chem. 1956 Apr;219(2):611–621. [PubMed] [Google Scholar]
  7. Hendrickson W. G., Kusano T., Yamaki H., Balakrishnan R., King M., Murchie J., Schaechter M. Binding of the origin of replication of Escherichia coli to the outer membrane. Cell. 1982 Oct;30(3):915–923. doi: 10.1016/0092-8674(82)90296-3. [DOI] [PubMed] [Google Scholar]
  8. Hirst T. R., Randall L. L., Hardy S. J. Cellular location of heat-labile enterotoxin in Escherichia coli. J Bacteriol. 1984 Feb;157(2):637–642. doi: 10.1128/jb.157.2.637-642.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Itikawa H., Ryu J. Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B. J Bacteriol. 1979 May;138(2):339–344. doi: 10.1128/jb.138.2.339-344.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jacq A., Kohiyama M. A DNA-binding protein specific for the early replicated region of the chromosome obtained from Escherichia coli membrane fractions. Eur J Biochem. 1980 Mar;105(1):25–31. doi: 10.1111/j.1432-1033.1980.tb04470.x. [DOI] [PubMed] [Google Scholar]
  11. 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]
  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. Messer W., Meijer M., Bergmans H. E., Hansen F. G., von Meyenburg K., Beck E., Schaller H. Origin of replication, oriC, of the Escherichia coli K12 chromosome: nucleotide sequence. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):139–145. doi: 10.1101/sqb.1979.043.01.020. [DOI] [PubMed] [Google Scholar]
  15. Moeremans M., De Raeymaeker M., Daneels G., De Mey J. FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes. Anal Biochem. 1986 Feb 15;153(1):18–22. doi: 10.1016/0003-2697(86)90054-0. [DOI] [PubMed] [Google Scholar]
  16. Oka A., Sugimoto K., Takanami M., Hirota Y. Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication. Mol Gen Genet. 1980 Apr;178(1):9–20. doi: 10.1007/BF00267207. [DOI] [PubMed] [Google Scholar]
  17. Sharma V., Farooqui S., Thind S. K., Nath R. Intestinal oxalate uptake in castrated male and female rats: evidence for altered brush border membrane composition. Biochem Int. 1984 Jul;9(1):69–75. [PubMed] [Google Scholar]
  18. 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]
  19. Tsugita A., Scheffler J. J. A rapid method for acid hydrolysis of protein with a mixture of trifluoroacetic acid and hydrochloric acid. Eur J Biochem. 1982 Jun;124(3):585–588. doi: 10.1111/j.1432-1033.1982.tb06634.x. [DOI] [PubMed] [Google Scholar]
  20. Zylicz M., Nieradko J., Taylor K. Escherichia coli dnaJ- and dnaK-gene products: synthesis in minicells and membrane-affinity. Biochem Biophys Res Commun. 1983 Jan 14;110(1):176–180. doi: 10.1016/0006-291x(83)91276-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES