Molecular and General Genetics MGG

, Volume 226, Issue 1–2, pp 190–197

The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins

  • Beate Rothe
  • Bernd Rothe
  • Peter Roggentin
  • Roland Schauer


An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.

Key words

Sialidase Gene structure Amino acid sequence Homology Clostridium septicum 


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  1. Berry AM, Paton JC, Glare EM, Hansmann D, Catcheside DEA (1988) Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli. Gene 71:299–305Google Scholar
  2. Calza R, Huttner E, Vincentz M, Rouze P, Calangau F, Vaucheret H, Cherel I, Meyer C, Kronenberger J, Caboche M (1987) Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants. Mol Gen Genet 209:552–562Google Scholar
  3. Cato EP, George WL, Finegold SM (1986) The clostridia. In: Sneath PHA (ed) Bergey's manual of systematic bacteriology, vol 2. Williams and Wilkins, Baltimore, pp 1141–1200Google Scholar
  4. Corfield AP, Schauer R (1982) Metabolism of sialic acids. In: Schauer R (ed) Sialic acids: chemistry, metabolism and function. (Cell biology monographs, vol 10) Springer, Wien, pp 195–262Google Scholar
  5. Davis LG, Dibney MD, Battey JF (1986) Basic methods in molecular biology. Elsevier, New YorkGoogle Scholar
  6. Galen J, Fasano A, Ketley J, Richardson S (1990) The role of neuraminidase in Vibrio cholerae pathogenesis. Abstracts of the annual meeting of the American Society for Microbiology, Anaheim, p 41Google Scholar
  7. Hahn CS, Lustig S, Strauss EG, Strauss JH (1988) Western equine encephalitis virus is a recombinant virus. Proc Natl Acad Sci USA 85:5997–6001Google Scholar
  8. Harth G, Haidaris CG, So M (1987) Neuraminidase from Trypanosoma cruzi: analysis of enhanced expression of the enzyme in infectious forms. Proc Natl Acad Sci USA 84:8320–8324Google Scholar
  9. Karplus PA, Walsh KA, Herriott JR (1984) Amino acid sequence of spinach ferredoxin:NADP+ oxidoreductase. Biochemistry 23:6576–6583Google Scholar
  10. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132Google Scholar
  11. Linn FV, Ward H, Prioli R, Pereira MEA (1990) Identification of neuraminidase in Giardia lamblia. Abstracts of the annual meeting of the American Society for Microbiology, Anaheim, p 75Google Scholar
  12. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  13. Marmur J (1961) A procedure for the isolation of DNA from microorganisms. J Mol Biol 3:208–218Google Scholar
  14. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78Google Scholar
  15. Müller HE (1970) The in vitro identification and the in vivo action of Clostridium septicum neuraminidase. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg AN 1 Orig 215:530–534Google Scholar
  16. Oh S-J, Kim Y-C, Park Y-W, Min S-Y, Kim I-S, Kang H-S (1987) Complete nucleotide sequence of the penicillin G acylase gene and the flanking regions, and its expression in Escherichia coli. Gen 56:87–97Google Scholar
  17. Pereira MEA (1983) A developmentally regulated neuraminidase activity in Tiypanosoma cruzi. Science 219:1444–1446Google Scholar
  18. Roggentin P, Berg W, Schauer R (1987) Purification and characterization of sialidase from Clostridium sordellii G12. Glycoconjugate J 4:349–359Google Scholar
  19. Roggentin P, Gutschker-Gdaniec G, Hobrecht R, Schauer R (1988a) Early diagnosis of clostridial gas gangrene using sialidase antibodies. Clin Chim Acta 173:251–262Google Scholar
  20. Roggentin P, Rothe B, Lottspeich F, Schauer R (1988b) Cloning and sequencing of a Clostridium perfringens sialidase gene. FEBS Lett 238:31–34Google Scholar
  21. Roggentin P, Rothe B, Kaper JB, Galen J, Lawrisuk L, Vimr ER, Schauer R (1989) Conserved sequences in bacterial and viral sialidases. Glycoconjugate J 6:349–353Google Scholar
  22. Romeo JM, Esmon B, Zusman DR (1986) Nucleotide sequence of the myxobacterial hemagglutinin gene contains four homologous domains. Proc Natl Acad Sci USA 83:6332–6336Google Scholar
  23. Rosenberg A, Schengrund C-L (1976) Sialidases. In: Rosenberg A, Schengrund C-L (eds) Plenum Press, New York, pp 295–298Google Scholar
  24. Rothe B, Roggentin P, Frank R, Blöcker H, Schauer R (1989) Cloning, sequencing and expression of a sialidase gene from Clostridium sordellii G12. J Gen Microbiol 135:3087–3096Google Scholar
  25. Russo TA, Thompson JS, Godoy VG, Malamy MH (1990) Cloning and expression of the Bacteriodes fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli. J Bacteriol 172:2594–2600Google Scholar
  26. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Nail Acad Sci USA 74:5463–5467Google Scholar
  27. Schauer R (1983) Glycosidases with special reference to the pathophysiological role of sialidases. In: Popper H, Reutter W, Gudat F, Köttgen E (eds) Structural carbohydrates of the liver. MTP Press, Lancaster, pp 83–97Google Scholar
  28. Schauer R (1985) Sialic acids and their role as biological masks. Trends Biochem Sci 10:357–360Google Scholar
  29. Schauer R, Sander-Wewer M, Gutschker-Gdaniec GHM, Roggentin P, Randow EA, Hobrecht R (1985) Sialidase activity in the sera of patients and rabbits with clostridial myonecrosis. Clin Chim Acta 146:119–127Google Scholar
  30. Schörgendorfer K, Schwab H, Lafferty RM (1987) Nucleotide sequence of a cloned 2.5 kb PstI-EcoRI Bacillus subtilis DNA fragment coding for levanase. Nucleic Acids Res 15:9606Google Scholar
  31. Southern E (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  32. Takle GB, Young A, Snary D, Hudson L, Nicholls SC (1989) Cloning and expression of a trypomastigote-specific 85-kilodalton surface antigen gene from Trypanosoma cruzi. Mol Biochem Parasitol 37:57–64Google Scholar
  33. Taussig R, Carlson M (1983) Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res 11:1943–1954Google Scholar
  34. Teufel M, Roggentin P, Schauer R (1989) Properties of sialidase isolated from Actinomyces viscosus DSM 43798. Biol Chem Hoppe-Seyler 370:435–443Google Scholar
  35. Vimr ER, Lawrisuk L, Galen J, Kaper JB (1988) Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli. J Bacteriol 170:1495–1504Google Scholar
  36. Von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690Google Scholar
  37. Watson M (1984) Compilation of published signal sequences. Nucleic Acids Res 12:5145–5164Google Scholar
  38. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and bost strains: nucleotide sequences of the Ml3mp18 and pUC19 vectors. Gene 33:103–119Google Scholar
  39. Young M, Minton NP, Staudenbauer WL (1989) Recent advances in the genetics of the clostridia. FEMS Microbiol Rev 63:301–326Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Beate Rothe
    • 1
  • Bernd Rothe
    • 2
  • Peter Roggentin
    • 1
  • Roland Schauer
    • 1
  1. 1.Biochemisches InstitutChristian-Albrechts-Universität zu KielKielGermany
  2. 2.Institut für MikrobiologieUniversität StuttgartStuttgart 1Germany

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