Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Mar;175(5):1293–1302. doi: 10.1128/jb.175.5.1293-1302.1993

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component.

W K Wang 1, K Kruus 1, J H Wu 1
PMCID: PMC193214  PMID: 8444792

Abstract

Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.

Full text

PDF
1293

Images in this article

1295Image
on p.1295

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Bagnara-Tardif C., Gaudin C., Belaich A., Hoest P., Citard T., Belaich J. P. Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum. Gene. 1992 Sep 21;119(1):17–28. doi: 10.1016/0378-1119(92)90062-t. [DOI] [PubMed] [Google Scholar]
  3. Béguin P., Cornet P., Aubert J. P. Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. J Bacteriol. 1985 Apr;162(1):102–105. doi: 10.1128/jb.162.1.102-105.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caruthers M. H. Gene synthesis machines: DNA chemistry and its uses. Science. 1985 Oct 18;230(4723):281–285. doi: 10.1126/science.3863253. [DOI] [PubMed] [Google Scholar]
  5. Coughlan M. P., Hon-Nami K., Hon-Nami H., Ljungdahl L. G., Paulin J. J., Rigsby W. E. The cellulolytic enzyme complex of Clostridium thermocellum is very large. Biochem Biophys Res Commun. 1985 Jul 31;130(2):904–909. doi: 10.1016/0006-291x(85)90502-9. [DOI] [PubMed] [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Foong F., Hamamoto T., Shoseyov O., Doi R. H. Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans. J Gen Microbiol. 1991 Jul;137(7):1729–1736. doi: 10.1099/00221287-137-7-1729. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Gräbnitz F., Rücknagel K. P., Seiss M., Staudenbauer W. L. Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases. Mol Gen Genet. 1989 May;217(1):70–76. doi: 10.1007/BF00330944. [DOI] [PubMed] [Google Scholar]
  11. Gräbnitz F., Seiss M., Rücknagel K. P., Staudenbauer W. L. Structure of the beta-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem. 1991 Sep 1;200(2):301–309. doi: 10.1111/j.1432-1033.1991.tb16186.x. [DOI] [PubMed] [Google Scholar]
  12. Grépinet O., Béguin P. Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B. Nucleic Acids Res. 1986 Feb 25;14(4):1791–1799. doi: 10.1093/nar/14.4.1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grépinet O., Chebrou M. C., Béguin P. Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum. J Bacteriol. 1988 Oct;170(10):4582–4588. doi: 10.1128/jb.170.10.4582-4588.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hall J., Hazlewood G. P., Barker P. J., Gilbert H. J. Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity. Gene. 1988 Sep 15;69(1):29–38. doi: 10.1016/0378-1119(88)90375-7. [DOI] [PubMed] [Google Scholar]
  15. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  16. Johnson E. A., Madia A., Demain A. L. Chemically Defined Minimal Medium for Growth of the Anaerobic Cellulolytic Thermophile Clostridium thermocellum. Appl Environ Microbiol. 1981 Apr;41(4):1060–1062. doi: 10.1128/aem.41.4.1060-1062.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnson E. A., Sakajoh M., Halliwell G., Madia A., Demain A. L. Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum. Appl Environ Microbiol. 1982 May;43(5):1125–1132. doi: 10.1128/aem.43.5.1125-1132.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Joliff G., Béguin P., Aubert J. P. Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum. Nucleic Acids Res. 1986 Nov 11;14(21):8605–8613. doi: 10.1093/nar/14.21.8605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kohring S., Wiegel J., Mayer F. Subunit Composition and Glycosidic Activities of the Cellulase Complex from Clostridium thermocellum JW20. Appl Environ Microbiol. 1990 Dec;56(12):3798–3804. doi: 10.1128/aem.56.12.3798-3804.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. LeGendre N., Matsudaira P. Direct protein microsequencing from Immobilon-P Transfer Membrane. Biotechniques. 1988 Feb;6(2):154–159. [PubMed] [Google Scholar]
  23. Losick R., Pero J. Cascades of Sigma factors. Cell. 1981 Sep;25(3):582–584. doi: 10.1016/0092-8674(81)90164-1. [DOI] [PubMed] [Google Scholar]
  24. Lüthi E., Jasmat N. B., Grayling R. A., Love D. R., Bergquist P. L. Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum". Appl Environ Microbiol. 1991 Mar;57(3):694–700. doi: 10.1128/aem.57.3.694-700.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martin F. H., Castro M. M., Aboul-ela F., Tinoco I., Jr Base pairing involving deoxyinosine: implications for probe design. Nucleic Acids Res. 1985 Dec 20;13(24):8927–8938. doi: 10.1093/nar/13.24.8927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  27. Matsushita O., Russell J. B., Wilson D. B. A Bacteroides ruminicola 1,4-beta-D-endoglucanase is encoded in two reading frames. J Bacteriol. 1991 Nov;173(21):6919–6926. doi: 10.1128/jb.173.21.6919-6926.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mayer F., Coughlan M. P., Mori Y., Ljungdahl L. G. Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy. Appl Environ Microbiol. 1987 Dec;53(12):2785–2792. doi: 10.1128/aem.53.12.2785-2792.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Navarro A., Chebrou M. C., Béguin P., Aubert J. P. Nucleotide sequence of the cellulase gene celF of Clostridium thermocellum. Res Microbiol. 1991 Nov-Dec;142(9):927–936. doi: 10.1016/0923-2508(91)90002-r. [DOI] [PubMed] [Google Scholar]
  31. Ng T. K., Zeikus J. G. Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414. Appl Environ Microbiol. 1981 Aug;42(2):231–240. doi: 10.1128/aem.42.2.231-240.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Reddy S. V., Hamsabhushanam K., Jagadeeswaran P. A rapid method for large-scale isolation of plasmid DNA by boiling in a plastic bag. Biotechniques. 1989 Sep;7(8):820–822. [PubMed] [Google Scholar]
  34. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Romaniec M. P., Clarke N. G., Hazlewood G. P. Molecular cloning of Clostridium thermocellum DNA and the expression of further novel endo-beta-1,4-glucanase genes in Escherichia coli. J Gen Microbiol. 1987 May;133(5):1297–1307. doi: 10.1099/00221287-133-5-1297. [DOI] [PubMed] [Google Scholar]
  36. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schwarz W. H., Schimming S., Rücknagel K. P., Burgschwaiger S., Kreil G., Staudenbauer W. L. Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum. Gene. 1988;63(1):23–30. doi: 10.1016/0378-1119(88)90542-2. [DOI] [PubMed] [Google Scholar]
  40. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  41. Shima S., Igarashi Y., Kodama T. Nucleotide sequence analysis of the endoglucanase-encoding gene, celCCD, of Clostridium cellulolyticum. Gene. 1991 Jul 31;104(1):33–38. doi: 10.1016/0378-1119(91)90461-j. [DOI] [PubMed] [Google Scholar]
  42. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  44. Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol. 1979;68:152–176. doi: 10.1016/0076-6879(79)68011-4. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yagüe E., Béguin P., Aubert J. P. Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum. Gene. 1990 Apr 30;89(1):61–67. doi: 10.1016/0378-1119(90)90206-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES