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. 1991 Nov;57(11):3062–3069. doi: 10.1128/aem.57.11.3062-3069.1991

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

M K Yeung 1, S R Fernandez 1
PMCID: PMC183928  PMID: 1781674

Abstract

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.

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  1. AMINOFF D. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J. 1961 Nov;81:384–392. doi: 10.1042/bj0810384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  3. Beighton D., Whiley R. A. Sialidase activity of the "Streptococcus milleri group" and other viridans group streptococci. J Clin Microbiol. 1990 Jun;28(6):1431–1433. doi: 10.1128/jcm.28.6.1431-1433.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brennan M. J., Cisar J. O., Sandberg A. L. A 160-kilodalton epithelial cell surface glycoprotein recognized by plant lectins that inhibit the adherence of Actinomyces naeslundii. Infect Immun. 1986 Jun;52(3):840–845. doi: 10.1128/iai.52.3.840-845.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brennan M. J., Cisar J. O., Vatter A. E., Sandberg A. L. Lectin-dependent attachment of Actinomyces naeslundii to receptors on epithelial cells. Infect Immun. 1984 Nov;46(2):459–464. doi: 10.1128/iai.46.2.459-464.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cassidy J. T., Jourdian G. W., Roseman S. The sialic acids. VI. Purification and properties of sialidase from Clostridium perfringens. J Biol Chem. 1965 Sep;240(9):3501–3506. [PubMed] [Google Scholar]
  8. Cisar J. O., Barsumian E. L., Curl S. H., Vatter A. E., Sandberg A. L., Siraganian R. P. Detection and localization of a lectin on Actinomyces viscosus T14V by monoclonal antibodies. J Immunol. 1981 Oct;127(4):1318–1322. [PubMed] [Google Scholar]
  9. Cisar J. O., David V. A., Curl S. H., Vatter A. E. Exclusive presence of lactose-sensitive fimbriae on a typical strain (WVU45) of Actinomyces naeslundii. Infect Immun. 1984 Nov;46(2):453–458. doi: 10.1128/iai.46.2.453-458.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  11. Corfield A. P., Veh R. W., Wember M., Michalski J. C., Schauer R. The release of N-acetyl- and N-glycolloyl-neuraminic acid from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidases. Biochem J. 1981 Aug 1;197(2):293–299. doi: 10.1042/bj1970293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Costello A. H., Cisar J. O., Kolenbrander P. E., Gabriel O. Neuraminidase-dependent hamagglutination of human erythrocytes by human strains of Actinomyces viscosus and Actinomyces naeslundii. Infect Immun. 1979 Nov;26(2):563–572. doi: 10.1128/iai.26.2.563-572.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drzeniek R., Scharmann W., Balke E. Neuraminidase and N-acetylneuraminate pyruvate-lyase of Pasteurella multocida. J Gen Microbiol. 1972 Sep;72(2):357–368. doi: 10.1099/00221287-72-2-357. [DOI] [PubMed] [Google Scholar]
  14. Ellen R. P., Fillery E. D., Chan K. H., Grove D. A. Sialidase-enhanced lectin-like mechanism for Actinomyces viscosus and Actinomyces naeslundii hemagglutination. Infect Immun. 1980 Feb;27(2):335–343. doi: 10.1128/iai.27.2.335-343.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flashner M., Wang P., Hurley J. B., Tanenbaum S. W. Properties of an inducible extracellular neuraminidase from an Arthrobacter isolate. J Bacteriol. 1977 Mar;129(3):1457–1465. doi: 10.1128/jb.129.3.1457-1465.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fraser A. G., Brown R. Neuraminidase production by Bacteroidaceae. J Med Microbiol. 1981 Feb;14(1):63–76. doi: 10.1099/00222615-14-1-63. [DOI] [PubMed] [Google Scholar]
  17. GIBBONS R. A. THE SENSITIVITY OF THE NEURAMINOSIDIC LINKAGE IN MUCOSUBSTANCES TOWARDS ACID AND TOWARDS NEURAMINIDASE. Biochem J. 1963 Nov;89:380–391. doi: 10.1042/bj0890380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kunimoto S., Aoyagi T., Takeuchi T., Umezawa H. Purification and characterization of Streptomyces sialidases. J Bacteriol. 1974 Aug;119(2):394–400. doi: 10.1128/jb.119.2.394-400.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Moncla B. J., Braham P. Detection of sialidase (neuraminidase) activity in Actinomyces species by using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid in a filter paper spot test. J Clin Microbiol. 1989 Jan;27(1):182–184. doi: 10.1128/jcm.27.1.182-184.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neuberger A., Ratcliffe W. A. The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm-Horsfall glycoprotein. Biochem J. 1972 Sep;129(3):683–693. doi: 10.1042/bj1290683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pardoe G. I. The inducible neuraminidase (N-acyl-neuraminyl hydrolase EC 3.2.1.18) of Klebsiella aerogenes NCIB 9479. Pathol Microbiol (Basel) 1970;35(6):361–376. doi: 10.1159/000162248. [DOI] [PubMed] [Google Scholar]
  23. Perlitsh M. J., Glickman I. Salivary neuraminidase. I. The presence of neuraminidase in human saliva. J Periodontol. 1966 Sep-Oct;37(5):368–373. doi: 10.1902/jop.1966.37.5.368. [DOI] [PubMed] [Google Scholar]
  24. Potier M., Mameli L., Bélisle M., Dallaire L., Melançon S. B. Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate. Anal Biochem. 1979 Apr 15;94(2):287–296. doi: 10.1016/0003-2697(79)90362-2. [DOI] [PubMed] [Google Scholar]
  25. Rogers R., Newbrun E., Tatevossian A. Neuraminidase activity in human dental plaque fluid. Arch Oral Biol. 1979;24(9):703–705. doi: 10.1016/0003-9969(79)90121-3. [DOI] [PubMed] [Google Scholar]
  26. Russo T. A., Thompson J. S., Godoy V. G., Malamy M. H. Cloning and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli. J Bacteriol. 1990 May;172(5):2594–2600. doi: 10.1128/jb.172.5.2594-2600.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sandberg A. L., Mudrick L. L., Cisar J. O., Brennan M. J., Mergenhagen S. E., Vatter A. E. Type 2 fimbrial lectin-mediated phagocytosis of oral Actinomyces spp. by polymorphonuclear leukocytes. Infect Immun. 1986 Nov;54(2):472–476. doi: 10.1128/iai.54.2.472-476.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Snyder M., Elledge S., Sweetser D., Young R. A., Davis R. W. Lambda gt 11: gene isolation with antibody probes and other applications. Methods Enzymol. 1987;154:107–128. doi: 10.1016/0076-6879(87)54073-3. [DOI] [PubMed] [Google Scholar]
  30. Teufel M., Roggentin P., Schauer R. Properties of sialidase isolated from Actinomyces viscosus DSM 43798. Biol Chem Hoppe Seyler. 1989 May;370(5):435–443. doi: 10.1515/bchm3.1989.370.1.435. [DOI] [PubMed] [Google Scholar]
  31. Tuyau J. E., Sims W. Occurrence of haemophili in dental plaque and their association with neuraminidase activity. J Dent Res. 1975 Jul-Aug;54(4):737–739. doi: 10.1177/00220345750540040701. [DOI] [PubMed] [Google Scholar]
  32. Uchida Y., Tsukada Y., Sugimori T. Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. J Biochem. 1979 Nov;86(5):1573–1585. doi: 10.1093/oxfordjournals.jbchem.a132675. [DOI] [PubMed] [Google Scholar]
  33. Vimr E. R., Lawrisuk L., Galen J., Kaper J. B. Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli. J Bacteriol. 1988 Apr;170(4):1495–1504. doi: 10.1128/jb.170.4.1495-1504.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yeung M. K., Chassy B. M., Cisar J. O. Cloning and expression of a type 1 fimbrial subunit of Actinomyces viscosus T14V. J Bacteriol. 1987 Apr;169(4):1678–1683. doi: 10.1128/jb.169.4.1678-1683.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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