Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Sep;61(9):3304–3310. doi: 10.1128/aem.61.9.3304-3310.1995

Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis.

D Geelen 1, M van Montagu 1, M Holsters 1
PMCID: PMC167611  PMID: 7574641

Abstract

Azorhizobium caulinodans ORS571, a symbiont of the tropical leguminous plant Sesbania rostrata, showed low, constitutive levels of endoglucanase (Egl) activity. A clone carrying the gene responsible for this phenotype was isolated via introduction of a genomic library into the wild-type strain and screening for transconjugants with enhanced Egl activity. By subcloning and expression in Escherichia coli, the Egl phenotype was allocated to a 3-kb EcoRI-BamHI fragment. However, sequence analysis showed the egl gene to be much larger, consisting of an open reading frame of 1,836 amino acids. Within the deduced polypeptide, three kinds of putative domains were identified: a catalytic domain, two cellulose-binding domains, and an eightfold reiterated motif. The catalytic domain belongs to the family A of cellulases. A C-terminal stretch of 100 amino acids was similar to family II cellulose-binding domains. A second copy of this domain occurred near the middle of the polypeptide, flanked by reiterated motifs. ORS571 mutants carrying a Tn5 insertion in the egl gene had lost the Egl activity. These mutants as well as Egl-overproducing strains showed a normal nodulation behavior, indistinguishable from wild-type nodulation on Sesbania rostrata under laboratory conditions.

Full Text

The Full Text of this article is available as a PDF (507.9 KB).

Selected References

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

  1. Baird S. D., Johnson D. A., Seligy V. L. Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans. J Bacteriol. 1990 Mar;172(3):1576–1586. doi: 10.1128/jb.172.3.1576-1586.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayer E. A., Morag E., Lamed R. The cellulosome--a treasure-trove for biotechnology. Trends Biotechnol. 1994 Sep;12(9):379–386. doi: 10.1016/0167-7799(94)90039-6. [DOI] [PubMed] [Google Scholar]
  3. Berger E., Jones W. A., Jones D. T., Woods D. R. Cloning and sequencing of an endoglucanase (end1) gene from Butyrivibrio fibrisolvens H17c. Mol Gen Genet. 1989 Oct;219(1-2):193–198. doi: 10.1007/BF00261176. [DOI] [PubMed] [Google Scholar]
  4. Biely P., Mislovicová D., Toman R. Soluble chromogenic substrates for the assay of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases. Anal Biochem. 1985 Jan;144(1):142–146. doi: 10.1016/0003-2697(85)90095-8. [DOI] [PubMed] [Google Scholar]
  5. Béguin P., Aubert J. P. The biological degradation of cellulose. FEMS Microbiol Rev. 1994 Jan;13(1):25–58. doi: 10.1111/j.1574-6976.1994.tb00033.x. [DOI] [PubMed] [Google Scholar]
  6. Carder J. H. Detection and quantitation of cellulase by Congo red staining of substrates in a cup-plate diffusion assay. Anal Biochem. 1986 Feb 15;153(1):75–79. doi: 10.1016/0003-2697(86)90063-1. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  8. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goethals K., Gao M., Tomekpe K., Van Montagu M., Holsters M. Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: nucleotide sequence and plant-inducible expression. Mol Gen Genet. 1989 Oct;219(1-2):289–298. doi: 10.1007/BF00261190. [DOI] [PubMed] [Google Scholar]
  10. Goethals K., Van den Eeede G., Van Montagu M., Holsters M. Identification and characterization of a functional nodD gene in Azorhizobium caulinodans ORS571. J Bacteriol. 1990 May;172(5):2658–2666. doi: 10.1128/jb.172.5.2658-2666.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gough C. L., Dow J. M., Keen J., Henrissat B., Daniels M. J. Nucleotide sequence of the engXCA gene encoding the major endoglucanase of Xanthomonas campestris pv. campestris. Gene. 1990 Apr 30;89(1):53–59. doi: 10.1016/0378-1119(90)90205-6. [DOI] [PubMed] [Google Scholar]
  12. Hall J., Hazlewood G. P., Huskisson N. S., Durrant A. J., Gilbert H. J. Conserved serine-rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing. Mol Microbiol. 1989 Sep;3(9):1211–1219. doi: 10.1111/j.1365-2958.1989.tb00271.x. [DOI] [PubMed] [Google Scholar]
  13. Henrissat B., Claeyssens M., Tomme P., Lemesle L., Mornon J. P. Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989 Sep 1;81(1):83–95. doi: 10.1016/0378-1119(89)90339-9. [DOI] [PubMed] [Google Scholar]
  14. Higashi S., Abe M. Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover. Appl Environ Microbiol. 1980 Dec;40(6):1094–1099. doi: 10.1128/aem.40.6.1094-1099.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hubbell D. H., Morales V. M., Umali-Garcia M. Pectolytic enzymes in Rhizobium. Appl Environ Microbiol. 1978 Jan;35(1):210–213. doi: 10.1128/aem.35.1.210-213.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Klyosov A. A. Trends in biochemistry and enzymology of cellulose degradation. Biochemistry. 1990 Nov 27;29(47):10577–10585. doi: 10.1021/bi00499a001. [DOI] [PubMed] [Google Scholar]
  17. Lao G., Ghangas G. S., Jung E. D., Wilson D. B. DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca. J Bacteriol. 1991 Jun;173(11):3397–3407. doi: 10.1128/jb.173.11.3397-3407.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Martinez-Molina E., Morales V. M., Hubbell D. H. Hydrolytic enzyme production by Rhizobium. Appl Environ Microbiol. 1979 Dec;38(6):1186–1188. doi: 10.1128/aem.38.6.1186-1188.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mateos P. F., Jimenez-Zurdo J. I., Chen J., Squartini A. S., Haack S. K., Martinez-Molina E., Hubbell D. H., Dazzo F. B. Cell-associated pectinolytic and cellulolytic enzymes in Rhizobium leguminosarum biovar trifolii. Appl Environ Microbiol. 1992 Jun;58(6):1816–1822. doi: 10.1128/aem.58.6.1816-1822.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Millward-Sadler S. J., Poole D. M., Henrissat B., Hazlewood G. P., Clarke J. H., Gilbert H. J. Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases. Mol Microbiol. 1994 Jan;11(2):375–382. doi: 10.1111/j.1365-2958.1994.tb00317.x. [DOI] [PubMed] [Google Scholar]
  21. Ndoye I., de Billy F., Vasse J., Dreyfus B., Truchet G. Root nodulation of Sesbania rostrata. J Bacteriol. 1994 Feb;176(4):1060–1068. doi: 10.1128/jb.176.4.1060-1068.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nicoll D. A., Longoni S., Philipson K. D. Molecular cloning and functional expression of the cardiac sarcolemmal Na(+)-Ca2+ exchanger. Science. 1990 Oct 26;250(4980):562–565. doi: 10.1126/science.1700476. [DOI] [PubMed] [Google Scholar]
  23. Reinhold-Hurek B., Hurek T., Claeyssens M., van Montagu M. Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph. J Bacteriol. 1993 Nov;175(21):7056–7065. doi: 10.1128/jb.175.21.7056-7065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roberts D. P., Denny T. P., Schell M. A. Cloning of the egl gene of Pseudomonas solanacearum and analysis of its role in phytopathogenicity. J Bacteriol. 1988 Apr;170(4):1445–1451. doi: 10.1128/jb.170.4.1445-1451.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ruvkun G. B., Sundaresan V., Ausubel F. M. Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes. Cell. 1982 Jun;29(2):551–559. doi: 10.1016/0092-8674(82)90171-4. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Tsien H. C., Dreyfus B. L., Schmidt E. L. Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata. J Bacteriol. 1983 Nov;156(2):888–897. doi: 10.1128/jb.156.2.888-897.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Van den Eede G., Deblaere R., Goethals K., Van Montagu M., Holsters M. Broad host range and promoter selection vectors for bacteria that interact with plants. Mol Plant Microbe Interact. 1992 May-Jun;5(3):228–234. doi: 10.1094/mpmi-5-228. [DOI] [PubMed] [Google Scholar]
  29. de Bruijn F. J., Lupski J. R. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids--a review. Gene. 1984 Feb;27(2):131–149. doi: 10.1016/0378-1119(84)90135-5. [DOI] [PubMed] [Google Scholar]
  30. van Brussel A. A., Bakhuizen R., van Spronsen P. C., Spaink H. P., Tak T., Lugtenberg B. J., Kijne J. W. Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium. Science. 1992 Jul 3;257(5066):70–72. doi: 10.1126/science.257.5066.70. [DOI] [PubMed] [Google Scholar]
  31. van Spronsen P. C., Bakhuizen R., van Brussel A. A., Kijne J. W. Cell wall degradation during infection thread formation by the root nodule bacterium Rhizobium leguminosarum is a two-step process. Eur J Cell Biol. 1994 Jun;64(1):88–94. [PubMed] [Google Scholar]
  32. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES