1887

Abstract

The SRT4 gene encoding levansucrase (EC 2.4.1.10) () was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the gene from the promoter in resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria and , respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the levansucrase with those of the levansucrase suggested that one of these motifs may be involved in the specificity of the synthesized product. Disruption of the gene in the genome of resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of .

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1996-05-01
2021-10-27
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References

  1. Altshul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J Mol Biol 1990; 215:403–410
    [Google Scholar]
  2. Alvarez B., Martinez-Drets G. Metabolic characterization of Acetobacter diayotrophicus. Can J Microbiol 1995; 41:918–924
    [Google Scholar]
  3. Bauw G., Van Damme J., Puype M., Vandekerckhove J., Gesser B., Ratz G.P., Lauridsen J.B., Celis J.E. Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels. Proc Natl Acad Sci USA 1989; 86:7701–7705
    [Google Scholar]
  4. Bringas R., Ricardo R., Fernández De Cossío J., Ochagavía M.E., Suárez A., Rodríguez R. BioSOS: A program package for the analysis of biological sequences in the microcomputer. Biotecnol Apl 1992; 9:180–185
    [Google Scholar]
  5. Cavalcante V.A., Ddbereiner J. A new acid-tolerant nitrogen-fixation bacterium associated with sugarcane. Plant Soil 1988; 108:23–31
    [Google Scholar]
  6. Chambert R., Petit-Glatron M.F. Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site directed-mutagenesis. Biochem J 1991; 279:35–41
    [Google Scholar]
  7. Coego A., Ramirez R., Menéndez G., Arrieta J., Hernández L., Suarez V., Selman G., Pérez S. Isolation and characterization of nitrogen-fixing bacteria associated to sugarcane. Rev Latinoam Microbiol 1992; 34:189–195
    [Google Scholar]
  8. Crittenden R.G., Doelle H.W. Structural identification of oligosaccharides produced by Zymomonas mobilis levansucrase. Biotechnol Lett 1993; 15:1055–1060
    [Google Scholar]
  9. De Ley J., Swings J., Gossel F. Genus I Acetobacter Beijerinck 1898, 215AL. In Bergey’s Manual of Systematic Bacteriology 1984 Edited by Krieg N.R., Holt J.G. Baltimore: Williams & Wilkins; 4 pp 268–274
    [Google Scholar]
  10. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X.W., Finlay D.R., Guiney D., Helinski D.R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 1985; 13:149–153
    [Google Scholar]
  11. Dong Z.M., Canny M.J., McCully M.E., Roboredo M.R., Cabadilla C.F., Ortega E., Rodes R. A nitrogen-fixing endophyte of sugarcane stems - a new role for the apoplast. Plant Physiol 1994; 105:1139–1147
    [Google Scholar]
  12. Fukaya M., Takemura H., Okumura H., Kawamura Y., Horinouchi S., Beppu T. Cloning of genes responsible for acetic acid resistance in Acetobacter aceti. J Bacteriol 1990; 172:2096–2104
    [Google Scholar]
  13. Geier G., Geider K. Characterization and influence on virulence of the levansucrase gene from the fireblight pathogen Erwinia amylovora. Physiol Mol Plant Pathol 1993; 42:387–404
    [Google Scholar]
  14. Gillis M., Kersters K., Hoste B., Janssens D., Kroppenstedt R.M., Stephan M.P., Teixeira K.R.S., Ddbereiner J., Deley J. Acetobacter diazotrophicus sp nov, a nitrogen-fixing acetic acid bacterium associated with sugarcane. Int J Syst Bacteriol 1989; 39:361–364
    [Google Scholar]
  15. Von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 1986; 14:4683–4690
    [Google Scholar]
  16. Hernández L., Arrieta J., Menendez C., Vazquez R., Coego A., Suarez V., Selman G., Petit-Glatron M.F., Chambert R. Isolation and enzymatic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochem J 1995; 309:113–118
    [Google Scholar]
  17. Hettwer U., Gross M., Rudolph K. Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv phaseolicola. J Bacteriol 1995; 177:2834–2839
    [Google Scholar]
  18. Inoue T., Sunagawa M., Mori A., Imai C., Fukuda M., Tagaki M., Yano K. Cloning and sequencing of the gene encoding the 72 kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti. J Bacteriol 1989; 171:3115–3122
    [Google Scholar]
  19. James E.K., Reis V.M., Olivares F.L., Baldani J.I., Ddbereiner J. Infection of sugar cane by the nitrogen-fixing bacterium Acetobacter diazotrophicus. J Exp Bot 1994; 45:757–766
    [Google Scholar]
  20. Miller J.H. Experiments in Molecular Genetics 1972 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Paula M.A., Reis V.M., Ddbereiner J. Interactions of Glomus clarum with Acetobacter diazotrophicus in infection of sweet potato (Ipomea batatas), sugarcane (Saccharum spp) and sweet sorghum (Sorghum vulgare). Biol Fértil Soils 1991; 11:111–115
    [Google Scholar]
  22. Pearson W.R., Lipman D.J. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 1988; 85:2444–2448
    [Google Scholar]
  23. Rost B., Casadio R., Fariselli P., Sander C. Transmembrane helices predicted at 95% accuracy. Protein Sci 1995; 4:521–533
    [Google Scholar]
  24. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: a Laboratory Manual 1989 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463–5467
    [Google Scholar]
  26. Saxena I.M., Lin F.C., Brown R.M. Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum. Plant Mol Biol 1990; 15:673–683
    [Google Scholar]
  27. Shiroza T., Kuramitsu H.K. Sequence analysis of the Streptococcus mutans fructosyltransferase gene and flanking regions. J Bacteriol 1988; 170:810–816
    [Google Scholar]
  28. Simon R., Priefer U., Puhler A. Vector plasmids for in vivo and in vitro manipulations of Gram-negative bacteria. In Molecular Genetics of the Bacteria-Plant Interaction 1983 Edited by Piihler A. Berlin: Springer-Verlag; pp 98–106
    [Google Scholar]
  29. Song K.B., Joo H.K., Rhee S.K. Nucleotide sequence of levansucrase gene (levU) of Zymomonas mobilis ZM1 (ATCC10988). Biochim Biophys Acta 1993; 1173:320–324
    [Google Scholar]
  30. Steinmetz M., Le Coq D., Aymerich S., Gonzy-Tr6boul G., Gay P. The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control site. Mol Gen Genet 1985; 200:220–228
    [Google Scholar]
  31. Tamaki T., Fukaya M., Takemura H., Tayama K., Okumura H., Kawamura Y., Nishiyama M., Horinouchi S., Beppu T. Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes. Biochim Biophys Acta 1991; 1088:292–300
    [Google Scholar]
  32. Tang L.B., Lenstra R., Borchert T.V., Nagarajan V. Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens. Gene 1990; 96:89–93
    [Google Scholar]
  33. Vriend G. what if: a molecular modeling and drug design program. J Mol Graph 1990; 8:52–56
    [Google Scholar]
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