1887

Abstract

Summary: The bv. BAL fructokinase gene was isolated on a 2.4 kb HI fragment from the cosmid pLA72 by complementation analysis of the Tn5-induced mutant BAL79, and confirmed by hybridization analysis. The nucleotide sequence of the gene was found to contain an open reading frame consisting of 978 bp encoding 326 amino acids, which was then compared to known fructokinase sequences. The fructokinase gene was not contained in an operon and is encoded separately from other enzymes of carbohydrate metabolism. Its product is therefore assigned to the group I fructokinases. A putative promoter (TTGACA-NGTTGAT), ribosome-binding site and termination sequence were identified. The Frk protein contained several motifs conserved in other known fructokinase sequences, including an ATP-binding and a substrate-binding motif. The hydropathy plot derived from the gene sequence data revealed the fructokinase as a hydrophilic protein. The fructokinase protein was purified to electrophoretic homogeneity by a three-step method using chromatofocusing, affinity chromatography and gel filtration. Its purity was confirmed by SDSPAGE and it was visualized as a single band by silver staining. The N-terminal amino acid sequence of the purified fructokinase confirmed the proposed open reading frame of the gene. The purified fructokinase had a molecular mass of 36.5 kDa, pl of 4.65, pH activity range of 6.0-9.0 (maximum activity at pH 8.0) and a Mg requirement. It had a of 0.31 mM and a of 31 μmol fructose 6-phosphate (mg protein) min with fructose as substrate. The bv. BAL fructokinase was biochemically and molecularly similar to other bacterial fructokinases.

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1996-02-01
2021-10-26
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References

  1. d'Aubenton Carafa Y., Brody E., Thermes C. 1990; Prediction of rho-independent Escherichia coli transcriptional terminators. J Mol Biol 216:835–858
    [Google Scholar]
  2. Aulkemeyer P., Ebner R., Heilenmann G., Jahreis K., Schmid K., Wrieden S., Lengeler J. L. 1991; Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria. Mol Biol 5:2913–2922
    [Google Scholar]
  3. Bach M. K., Magee W. E., Bum's R. H. 1958; Translocation of photosynthetic products to soybean nodules and their role in nitrogen fixation. Plant Physiol 33:118–124
    [Google Scholar]
  4. Baysdorfer C, Kremer D. F., Sicher R. C. 1989; Partial purification and characterization of fructokinase activity from barley leaves. J Plant Physiol 134:156–161
    [Google Scholar]
  5. Bethlenfalvay G. J., Phillips D. A. 1977; Photosynthesis and symbiotic nitrogen fixation in Phaseolus vulgaris L. Genetic Engineering for Nitrogen Fixation401–408 Edited by Hollaender A. New York & London: Plenum Press;
    [Google Scholar]
  6. Blatch G. L., Woods D. R. 1991; Nucleotide sequence and analysis of the Vibrio alginolyticus scr repressor-encoding gene (scrR). Gene 1010:45–50
    [Google Scholar]
  7. Blatch G. L., Scholle R. S., Woods D. R. 1990; Nucleotide sequence and analysis of the Vibrio alginolyticus sucrose uptake-encoding region. Gene 95:17–23
    [Google Scholar]
  8. Bradford M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 92:248–254
    [Google Scholar]
  9. Brockman J., Heuel H., Lengeler J. W. 1992; Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132. Mol & Gen Genet 235:22–32
    [Google Scholar]
  10. Brune D. C. 1992; Alkylation of cysteine with acrylamide for protein sequence analysis. Anal Biochem 207:285–290
    [Google Scholar]
  11. Chein A., Edgar D. B., Trela J. M. 1976; Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 121:1550–1557
    [Google Scholar]
  12. Cowan P. J., Nagesha H., Leonard L., Howard J. L., Pittard A. J. 1991; Characterization of the major promoter for the plasmid-encoded sucrose genes scrY, scr A and scrB. J Bacteriol 173:7464–7470
    [Google Scholar]
  13. Curling J. M. 1970; The use of Sephadex in the separation, purification and characterization of biological materials. Exp Physiol Biochem 3:417–484
    [Google Scholar]
  14. Dazzo F. B., Hubbel D. H. 1975; Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizpbium-clover association. Appl Microbiol 30:1017–1033
    [Google Scholar]
  15. Dean P. D. G., Watson D. H. 1979; Protein purification using immobilized triazine dyes. J Chromatogr 165:301–319
    [Google Scholar]
  16. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
    [Google Scholar]
  17. Ditta G., Stanfield S., Corbin D., Helinski D. R. 1980; Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene clone bank of Rhizobium meliloti. Proc Natl AcadSci USA 77:7347–7351
    [Google Scholar]
  18. Ditta G., Schimhauser T, Yakobson E., Lu P., Lang X. W., Finlay D. R., Guiney D., Helinski D. 1985; Plasmids related to the broad host range vector pRK290 useful for gene cloning and for monitoring gene expression. Plasmid 13:149–153
    [Google Scholar]
  19. Duncan M. 1981; Properties of Tn5-induced carbohydrate mutants of Rhizobium meliloti. J Gen Microbiol 122:61–67
    [Google Scholar]
  20. French B. A., Chang S. H. 1987; Nucleotide sequence of the phosphofructokinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene. Gene 54:65–71
    [Google Scholar]
  21. Glenn A. R., Arwas R., McKay I. A., Di Iworth M. J. 1984; Fructose metabolism in wild-type, frucktokinase-negative and revertant strains of Rhizobium leguminosarum. J Gen Microbiol 119:267–271
    [Google Scholar]
  22. Hanahan D. 1983; Studies on transformation of E. coli with plasmids. J Mol Biol 166:557–580
    [Google Scholar]
  23. Hardesty C, Ferran C., DiRienzo J. M. 1991; Plasmid-mediated sucrose metabolism in Escherichia coli: characterization of scrY, the structural gene for a phosphoenolpyruvate-dependent sucrose phosphotransferase system outer membrane porin. J Bacteriol 173:449–456
    [Google Scholar]
  24. Harper D. 1981; Isoelectric focusing in agarose gels. Electrophoresis ‘81, Proceedings of the 3rd Conference on Electrophoresis205–212 Edited by Allen R. C., Arnaud P. New York: Walter deGruyter;
    [Google Scholar]
  25. Heinisch J., Ritzel R. G., von Borstel R. C., Aquilera A., Rodico R., Zimmermann F. K. 1989; The phosphofructokinase genes of yeast evolved from two duplication events. Gene 78:309–321
    [Google Scholar]
  26. Hope J. N., Bell A. W., Hermodson M. A., Groarke J. M. 1986; Ribokinase from Escherichia coli K12: nucleotide sequence and overexpression of the rbsK gene and purification of ribokinase. J Biol Chem 261:7663–7668
    [Google Scholar]
  27. Jahreis K., Lengeler J. W. 1993; Molecular analysis of two ScrR repressors and of a ScrR-FruR hydrid repressor for sucrose and d-fructose specific regulons from enteric bacteria. Mol Microbiol 9:195–209
    [Google Scholar]
  28. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
    [Google Scholar]
  29. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  30. Lallan G. 1990; Chromatofocusing. Guide To Protein Purification380–392 Edited by Deutscher M. P. San Diego: Academic Press;
    [Google Scholar]
  31. Leary J. J., Brigati D. J., Ward D. C. 1983; Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bioblots. Proc Natl Acad Sci USA 80:4045–4049
    [Google Scholar]
  32. Lineweaver H., Burke D. 1934; The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666
    [Google Scholar]
  33. Mandal N. C., Chakrabartty P. N. 1993; Succinate-mediated catabolite repression of enzymes of glucose metabolism in root-nodule bacteria. Curr Microbiol 26:247–251
    [Google Scholar]
  34. Maniatis T., Fritsch E. F., Sambrook J. 1989 Molecular Cloning: a Laboratory Manual Cold Spring Harbor N.Y.: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Martinez deDrets G., Arias A. 1970; Metabolism of some polyols by Rhizobium meliloti. J Bacteriol 103:97–103
    [Google Scholar]
  36. McLaughlin R. E. 1989 PhD thesis Clemson University;
  37. McLaughlin R. E., Hughes T. A. 1989; Transposon mutagenesis and complementation of the frucktokinase gene in Rhizobium leguminosarum biovar trifolii. J Gen Microbiol 135:2329–2334
    [Google Scholar]
  38. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. 1981; Ultrasensitive stain for proteins: polyacrylamide gels show regional variation in cerebrospinal fluid. Science 211:1437
    [Google Scholar]
  39. O'Neill M. C. 1989; Escherichia coli promoters. I. Consensus as it relates to spacing class, specificity, repeat substructure, and three-dimensional organization. J Biol Chem 264:5522–5530
    [Google Scholar]
  40. Osteras M., Finan T. M., Stanley J. 1991; Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host dependent symbiotic phenotype. Mol & Gen Genet 230:257–269
    [Google Scholar]
  41. Pate J. S. 1977; Functional biology of dinitrogen fixation by legumes. A Treatise on Dinitrogen Fixation, Section III. Biology473–518 Edited by Hardy R. W. F., Silver W. S. New York: Wiley-Interscience;
    [Google Scholar]
  42. Podkovyrov M. P., Zeikus J. G. 1993; Purification and characterization of phosphoenolpyruvate carboxykinase, a catalytic CO2-fixing enzyme, from Anaerobiospirillum succiniciproducens. J Gen Microbiol 139:223–228
    [Google Scholar]
  43. Ronson C. W., Primrose S. B. 1979; Carbohydrate metabolism in Rhizobium trifolii: identification and symbiotic properties of mutants. J Gen Microbiol 112:77–88
    [Google Scholar]
  44. Sabater B., Delafuente G. 1975; Kinetic properties and related changes of molecular weight in a fructokinase from Streptomyces violaceoruber. Biochim Biophys Acta 377:258–270
    [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 78:4284–4288
    [Google Scholar]
  46. Sato Y., Yamamato Y., Kizaki H., Kuramitsu H. K. 1993; Isolation, characterization and sequence analysis of the scrK gene encoding fructokinase of Streptococcus mutans. J Gen Microbiol 139:921–927
    [Google Scholar]
  47. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
    [Google Scholar]
  48. Stormo G. T., Scheider T. D., Gold L. M. 1982; Characterization of translational initiation sites in E. coli. Nucleic Acids Res 10:2991–2996
    [Google Scholar]
  49. Stowers M. D., Elkan G. H. 1985; Regulation of hexose catabolism in Rhizobium sp. 32H1. FEMS Microbiol Lett 26:45–48
    [Google Scholar]
  50. Thompson J., Sackett D. L., Donkersloot J. A. 1991; Purification and properties of frucktokinase I from Lactococcus lactis. J Biol Chem 266:22626–22633
    [Google Scholar]
  51. Thompson J., Nguyen N. Y., Robrish S. A. 1992; Sucrose fermentation by Fusobacterium mortiferum ATCC 25557: transport, catabolism and products. J Bacteriol 174:3227–3235
    [Google Scholar]
  52. Ucker D. S., Signer E. R. 1978; Catabolite-repression-like phenomenon in Rhizobium meliloti. J Bacteriol 136:1197–1200
    [Google Scholar]
  53. Vincent J. M. 1970 A Manual for the Practical Study of Root-nodule Bacteria (International Biological Programme Handbook no. 15) Oxford: Blackwell Scientific Publications;
    [Google Scholar]
  54. Vretblad P. 1976; Purification of lectins by biospecific affinity chromatography. Biochim Biophys Acta 434:169–176
    [Google Scholar]
  55. Wu L, Reizer A., Reizer J., Cai B., Tomich J. M., Saier M. H. Jr 1991; Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinase of Escherichia coli. J Bacteriol 173:3317–3127
    [Google Scholar]
  56. Zembrzuski B., Chilco P., Liu X., Liu J., Conway T., Scopes R. 1992; Cloning, sequencing and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases. J Bacteriol 174:3455–3460
    [Google Scholar]
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