Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 May;176(10):2828–2834. doi: 10.1128/jb.176.10.2828-2834.1994

Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome.

S Salamitou 1, M Lemaire 1, T Fujino 1, H Ohayon 1, P Gounon 1, P Béguin 1, J P Aubert 1
PMCID: PMC205436  PMID: 8188584

Abstract

The ORF3 gene of Clostridium thermocellum encodes a polypeptide (ORF3p) which contains a receptor domain for the docking sequence borne by the catalytic subunits of the cellulosome and a triplicated domain related to some bacterial cell surface proteins. It was thus surmised that ORF3p is a surface protein. In this study, this hypothesis was confirmed. Subcellular fractionation, Western blotting (immunoblotting), and electron microscopy of immunocytochemically labeled cells indicated that ORF3p produced by C. thermocellum was located in the outer surface layer of the bacterium. This layer appeared to consist of a soft matrix shedding off particulate fragments. Nonsedimenting ORF3p derived from sonicated cells was associated with high-molecular-mass fractions (> 20 MDa), probably corresponding to fragments of the outer cell layer. The same high-molecular-mass fractions also contained the cellulosomal marker CipA. Contrary to CipA, however, ORF3p was not associated with 2- to 4-MDa fractions corresponding to individual cellulosomes, and a significant fraction of ORF3p failed to bind to cellulose. It is proposed that ORF3 and ORF3p be renamed olpA and OlpA, respectively (for outer layer protein).

Full text

PDF
2828

Images in this article

2829Image
on p.2829
2829Image
on p.2829
2830Image
on p.2830
2831Image
on p.2831
2832Image
on p.2832
2833Image
on p.2833

Selected References

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

  1. Bayer E. A., Lamed R. Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose. J Bacteriol. 1986 Sep;167(3):828–836. doi: 10.1128/jb.167.3.828-836.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayer E. A., Setter E., Lamed R. Organization and distribution of the cellulosome in Clostridium thermocellum. J Bacteriol. 1985 Aug;163(2):552–559. doi: 10.1128/jb.163.2.552-559.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fujino T., Béguin P., Aubert J. P. Cloning of a Clostridium thermocellum DNA fragment encoding polypeptides that bind the catalytic components of the cellulosome. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):165–170. doi: 10.1016/0378-1097(92)90602-k. [DOI] [PubMed] [Google Scholar]
  4. Fujino T., Béguin P., Aubert J. P. Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface. J Bacteriol. 1993 Apr;175(7):1891–1899. doi: 10.1128/jb.175.7.1891-1899.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gerngross U. T., Romaniec M. P., Kobayashi T., Huskisson N. S., Demain A. L. Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology. Mol Microbiol. 1993 Apr;8(2):325–334. doi: 10.1111/j.1365-2958.1993.tb01576.x. [DOI] [PubMed] [Google Scholar]
  6. Grépinet O., Chebrou M. C., Béguin P. Purification of Clostridium thermocellum xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C. thermocellum. J Bacteriol. 1988 Oct;170(10):4576–4581. doi: 10.1128/jb.170.10.4576-4581.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hobot J. A., Bjornsti M. A., Kellenberger E. Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution. J Bacteriol. 1987 May;169(5):2055–2062. doi: 10.1128/jb.169.5.2055-2062.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Lamed R., Setter E., Bayer E. A. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. J Bacteriol. 1983 Nov;156(2):828–836. doi: 10.1128/jb.156.2.828-836.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Salamitou S., Raynaud O., Lemaire M., Coughlan M., Béguin P., Aubert J. P. Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA. J Bacteriol. 1994 May;176(10):2822–2827. doi: 10.1128/jb.176.10.2822-2827.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Salamitou S., Tokatlidis K., Béguin P., Aubert J. P. Involvement of separate domains of the cellulosomal protein S1 of Clostridium thermocellum in binding to cellulose and in anchoring of catalytic subunits to the cellulosome. FEBS Lett. 1992 Jun 8;304(1):89–92. doi: 10.1016/0014-5793(92)80595-8. [DOI] [PubMed] [Google Scholar]
  13. Tailliez P., Girard H., Millet J., Beguin P. Enhanced Cellulose Fermentation by an Asporogenous and Ethanol-Tolerant Mutant of Clostridium thermocellum. Appl Environ Microbiol. 1989 Jan;55(1):207–211. doi: 10.1128/aem.55.1.207-211.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tokatlidis K., Salamitou S., Béguin P., Dhurjati P., Aubert J. P. Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components. FEBS Lett. 1991 Oct 21;291(2):185–188. doi: 10.1016/0014-5793(91)81279-h. [DOI] [PubMed] [Google Scholar]

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

RESOURCES