Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Feb;178(4):1200–1203. doi: 10.1128/jb.178.4.1200-1203.1996

Interactions of the CelS binding ligand with various receptor domains of the Clostridium thermocellum cellulosomal scaffolding protein, CipA.

B Lytle 1, C Myers 1, K Kruus 1, J H Wu 1
PMCID: PMC177785  PMID: 8576058

Abstract

The Clostridium thermocellum cellulosomal scaffolding protein, CipA, acts as an anchor on the cellulose surface for the various catalytic subunits of the cellulosome, a large extracellular cellulase complex. CipA contains nine repeated domains that serve as receptors for the cellulosomal catalytic subunits, each of which carries a conserved, duplicated ligand sequence (DS). Four representative CipA receptor domains with sequence dissimilarity were cloned and expressed in Escherichia coli. The interaction of these cloned receptor domains with the duplicated ligand sequence of CelS (expressed as a thioredoxin fusion protein, TRX-DSCelS), was studied by nondenaturing polyacrylamide gel electrophoresis. TRX-DSCelS formed a stable complex with each of the four receptor domains, indicating that CelS, the most abundant cellulosomal catalytic subunit, binds nonselectively to all of the CipA receptors. Conversely, the duplicated sequence of CipA (in the form of TRX-DSCipA), which is homologous to that of CelS, did not bind to any of the receptors under the experimental conditions.

Full Text

The Full Text of this article is available as a PDF (222.0 KB).

Selected References

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

  1. 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]
  2. 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]
  3. Kruus K., Lua A. C., Demain A. L., Wu J. H. The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9254–9258. doi: 10.1073/pnas.92.20.9254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. LaVallie E. R., DiBlasio E. A., Kovacic S., Grant K. L., Schendel P. F., McCoy J. M. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N Y) 1993 Feb;11(2):187–193. doi: 10.1038/nbt0293-187. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Morag E., Bayer E. A., Hazlewood G. P., Gilbert H. J., Lamed R. Cellulase Ss (CelS) is synonymous with the major cellobiohydrolase (subunit S8) from the cellulosome of Clostridium thermocellum. Appl Biochem Biotechnol. 1993 Nov;43(2):147–151. doi: 10.1007/BF02916439. [DOI] [PubMed] [Google Scholar]
  8. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Wang W. K., Kruus K., Wu J. H. Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component. J Bacteriol. 1993 Mar;175(5):1293–1302. doi: 10.1128/jb.175.5.1293-1302.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Wang W. K., Kruus K., Wu J. H. Cloning and expression of the Clostridium thermocellum celS gene in Escherichia coli. Appl Microbiol Biotechnol. 1994 Nov;42(2-3):346–352. doi: 10.1007/BF00902740. [DOI] [PubMed] [Google Scholar]
  13. Yaron S., Morag E., Bayer E. A., Lamed R., Shoham Y. Expression, purification and subunit-binding properties of cohesins 2 and 3 of the Clostridium thermocellum cellulosome. FEBS Lett. 1995 Feb 27;360(2):121–124. doi: 10.1016/0014-5793(95)00074-j. [DOI] [PubMed] [Google Scholar]

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

RESOURCES