Skip to main content
SpringerLink
Log in

Molecular cloning, expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and35S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley β-glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)-β-d-glucanase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Literature Cited

  1. Beguin P, Millet J, Chavaux S, Yague E, Aubert JP (1989) Genetics of bacterial cellulases. In: Coughlan MP (ed) Enzyme systems for lignocellulose degradation. London: Elsevier Applied Science, pp 57–72

    Google Scholar 

  2. Besle JM, Lassalas B, Thivend P (1981) Digestion des glucides cytoplasmiques de la féverole par le veau préruminant. Reprod Nutr Dev 21:629–649

    Google Scholar 

  3. Bethesda Research Laboratories (1986) BRLpUC host:E. coli DH5 & TM competent cells. Bethesda Res. Lab Focus 8 (2): 9

    Google Scholar 

  4. Cavicchioli R, Watson K (1991) Molecular cloning expression and characterization of endoglucanase genes fromFibrobacter succinogenes AR1. Appl Environ Microbiol 57:359–365

    Google Scholar 

  5. Cavicchioli R, East PD, Watson K (1991)End A Fs , a novel family E endoglucanase gene fromFibrobacter succinogenes AR1. J Bacteriol 173:3265–3268

    Google Scholar 

  6. Cheng KJ, Stewart CS, Dinsdale D, Costerton JW (1984) Electron microscopy of bacteria involved in the digestion of plant cell walls. Anim Feed Sci Technol 10:93–120

    Google Scholar 

  7. Crosby B, Collier B, Thomas DY, Teather M, Erfle JD (1984) Cloning and expression inE. coli of cellulase genes fromB. succinogenes. In: S. Hasnain (ed), Proc. 5th Can. Bioenergy R and D Semin. Barking, England: Elsevier Applied Science, pp 573–576

    Google Scholar 

  8. Dougan B, Kehoe M (1984) The minicell system as a method for studying expression from plasmid DNA. Methods Microbiol 17:233–258

    Google Scholar 

  9. Erfle JD, Teather RM, Wood PJ, Irvin JE (1988) Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d glucan-4-glucanohydrolase, EC 3.2.1.73) fromBacteroides succinogenes cloned inEscherichia coli. Biochem J 255:833–841

    Google Scholar 

  10. Forsberg CW, Beveridge TJ, Hellstrom A (1981) Cellulase and xylanase release fromBacteroides succinogenes and its importance in the rumen environment. Appl Environ Microbiol 42:886–896

    Google Scholar 

  11. Gaudet G (1987) Les produits de dégradation de la cellulose par les cellulases deBacteroides succinogenes, bactérie cellulolytique du rumen. Reprod Nutr Dev 27:239–240

    Google Scholar 

  12. Gaudet G, Forano E, Dauphin G, Delort AM (1992) Futile cycling of glycogen inFibrobacter succinogenes as shown by in situ1H-NMR and13C-NMR investigation. Eur J Biochem 207:155–162

    Google Scholar 

  13. Gong J, Lo RYC, Forsberg CW (1989) Molecular cloning and expression inEscherichia coli of a cellodextrinase gene fromBacteroides succinogenes S85. Appl Environ Microbiol 55:132–136

    Google Scholar 

  14. Groleau D, Forsberg CW (1981) The cellulolytic activity of the rumen bacteriumBacteroides succinogenes. Can J Microbiol 27:517–530

    Google Scholar 

  15. Groleau D, Forsberg CW (1983a) Partial characterization of the extracellular endoglucanase produced byBacteroides succinogenes. Can J Microbiol 29:504–517

    Google Scholar 

  16. Groleau D, Forsberg CW (1983b) Solubilization of the extracellular membranous carboxymethylcellulase ofBacteroides succinogenes by trypsin. Can J Microbiol 29:710–717

    Google Scholar 

  17. Hazlewood GP, Romaniec MPM, Davidson K, Grepinet O, Beguin P, Millet J, Raynaud O, Aubert JP (1988) A catalogue ofClostridium thermocellum endoglucanases, β-glucosidases and xylanases genes cloned inEscherichia coli. FEMS Microbiol Lett 51:231–236

    Google Scholar 

  18. Hespell RB (1990) Genetic manipulation of ruminal microorganisms. In: Akin DE, et al. (eds) Microbial and plant opportunities to improve lignocellulose utilization by ruminants. Elsevier Science Publishing Co., Inc., pp 343–355

  19. Hu YJ, Smith DC, Cheng KJ, Forsberg CW (1991) Cloning of a xylanase gene fromFibrobacter succinogenes 135 and its expression inEscherichia coli. Can J Microbiol 37:554–561

    Google Scholar 

  20. Huang L, Forsberg CW (1987) Isolation of a cellodextrinase fromBacteroides succinogenes. Appl Environ Microbiol 53:1034–1041

    Google Scholar 

  21. Huang L, Forberg CW (1988) Purification and comparison of periplasmic and extracellular forms of the cellodextrinase fromBacteroides succinogenes. Appl Environ Microbiol 54:1488–1493

    Google Scholar 

  22. Huang L, Forsberg CW, Thomas DY (1988) Purification and characterization of a chloride stimulated cellobiosidase fromBacteroides succinogenes. J. Bacteriol 170:2923–2932

    Google Scholar 

  23. Hungate RE (1966) The rumen and its microbes. New York: Academic press, Inc.

    Google Scholar 

  24. Irvin JE, Teather RM (1988) Cloning and expression of aBacteroides succinogenes mixed-linkage β-glucanase (1,3-1,4-β-d-glucan 4-glucanohydrolase) gene inEscherichia coli. Appl Environ Microbiol 54:2672–2676

    Google Scholar 

  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Google Scholar 

  26. Lowry OH, Rosenbrough NJ, Farr AJ, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193:267–275

    Google Scholar 

  27. Martin C, Boeuf C, Bousquet F (1991)Escherichia coli CS31A fimbriae: molecular cloning, expression and homology with the K88 determinant. Microb Pathogen 10:429–442

    Google Scholar 

  28. Matte A, Forsberg CW (1992) Purification, characterization, and mode of action of endoxylanases 1 and 2 fromFibrobacter succinogenes S85. Appl Environ Microbiol 58:157–168

    Google Scholar 

  29. McDermid KP, Forsberg CW, MacKenzie CR (1990) Purification and properties of an acetyl xylan esterase fromFibrobacter succinogenes S85. Appl Environ Microbiol 56:3805–3810

    Google Scholar 

  30. McGavin M, Forsberg CW (1988) Isolation and characterization of endoglucanase 1 and 2 fromBacteroides succinogenes S85. J Bacteriol 170:2914–2922

    Google Scholar 

  31. McGavin MJ, Forsberg CW, Crosby B, Bell AW, Dignard D, Thomas DY (1989) Structure of the cel-3 gene fromFibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3. J Bacteriol 171:5587–5595

    Google Scholar 

  32. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press

    Google Scholar 

  33. Oakley BR, Kirsh DR, Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105:361–363

    Google Scholar 

  34. Reeve JN (1972) Use of minicells for bacteriophage directed polypeptide synthesis. Methods Enzymol 68:493–503

    Google Scholar 

  35. Sambrook JS, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press

    Google Scholar 

  36. Sipat A, Taylor KA, Forsberg CW, Krell PJ (1987) Molecular cloning of a xylanase gene fromBacteroides succinogenes and its expression inEscherichia coli. Appl Environ Microbiol 53:477–481

    Google Scholar 

  37. Taylor KA, Crosby B, McGavin M, Forsberg CW, Thomas DY (1987) Characteristics of the endoglucanase encoded by a cel gene fromBacteroides succinogenes expressed inEscherichia coli. Appl Environ Microbiol 53:41–46

    Google Scholar 

  38. Teather RM (1990) Structure of β-glucanases genes from rumen bacteria. In: Akin DE, et al. (eds) Microbial and plant opportunities to improve lignocellulose utilization by ruminants. Elsevier Science Publishing Co., Inc., pp 377–388

  39. Teather RM, Erfle JD (1990) DNA sequence of aFibrobacter succinogenes mixed-linkage β-glucanase (1,3-1,4-β-d-glucan 4-glucanohydrolase) gene. J Bacteriol 172:3837–3841

    Google Scholar 

  40. Tommassen J, Lugtenberg B (1980) Outer membrane protein e ofEscherichia coli K12 is co-regulated with alkaline phosphatase. J Bacteriol 143:151–157

    Google Scholar 

  41. Wood TM (1970) The cellulase ofFusarium solani: purification and specificity of the β-(1,4)-glucanase and the β-d-glucosidase components. Biochem J 121:353–362

    Google Scholar 

  42. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Microbiology, INRA, CR de Clermont-Ferrand-Theix, 63122, Saint-Genès-Champanelle, France

    Evelyne Forano, Véronique Broussolle & Geneviève Gaudet

  2. University Center of Sciences and Techniques, Blaise Pascal University, Aubière, France

    Geneviève Gaudet

  3. Department of Biological Sciences, University of Exeter, Exeter, UK

    John A. Bryant

Authors
  1. Evelyne Forano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Véronique Broussolle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Geneviève Gaudet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John A. Bryant
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Forano, E., Broussolle, V., Gaudet, G. et al. Molecular cloning, expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85. Current Microbiology 28, 7–14 (1994). https://doi.org/10.1007/BF01575979

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01575979

Keywords

Search

Navigation