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. 1994 Jan;176(2):450–459. doi: 10.1128/jb.176.2.450-459.1994

Genetics of galactose metabolism of Erwinia amylovora and its influence on polysaccharide synthesis and virulence of the fire blight pathogen.

M Metzger 1, P Bellemann 1, P Bugert 1, K Geider 1
PMCID: PMC205069  PMID: 7507102

Abstract

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the galT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules of E. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact O antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.

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Selected References

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

  1. Adhya S., Miller W. Modulation of the two promoters of the galactose operon of Escherichia coli. Nature. 1979 Jun 7;279(5713):492–494. doi: 10.1038/279492a0. [DOI] [PubMed] [Google Scholar]
  2. Austin E. A., Graves J. F., Hite L. A., Parker C. T., Schnaitman C. A. Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus. J Bacteriol. 1990 Sep;172(9):5312–5325. doi: 10.1128/jb.172.9.5312-5325.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ayers A. R., Ayers S. B., Goodman R. N. Extracellular Polysaccharide of Erwinia amylovora: a Correlation with Virulence. Appl Environ Microbiol. 1979 Oct;38(4):659–666. doi: 10.1128/aem.38.4.659-666.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BUTTIN G. M'ECANISMES R'EGULATEURS DANS LA BIOSYNTH'ESE DES ENZYMES DU M'ETABOLISME DU GALACTOSE CHEZ ESCHERICHIA COLI K12. II. LE D'ETERMINISME G'EN'ETIQUE DE LA R'EGULATION. J Mol Biol. 1963 Aug;7:183–205. doi: 10.1016/s0022-2836(63)80045-5. [DOI] [PubMed] [Google Scholar]
  5. Barny M. A., Guinebretière M. H., Marçais B., Coissac E., Paulin J. P., Laurent J. Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence. Mol Microbiol. 1990 May;4(5):777–786. doi: 10.1111/j.1365-2958.1990.tb00648.x. [DOI] [PubMed] [Google Scholar]
  6. Bellemann P., Geider K. Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization. J Gen Microbiol. 1992 May;138(5):931–940. doi: 10.1099/00221287-138-5-931. [DOI] [PubMed] [Google Scholar]
  7. Bernhard F., Coplin D. L., Geider K. A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii. Mol Gen Genet. 1993 May;239(1-2):158–168. doi: 10.1007/BF00281614. [DOI] [PubMed] [Google Scholar]
  8. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  9. Buendia A. M., Enenkel B., Köplin R., Niehaus K., Arnold W., Pühler A. The Rhizobium meliloti exoZl exoB fragment of megaplasmid 2: ExoB functions as a UDP-glucose 4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM. Mol Microbiol. 1991 Jun;5(6):1519–1530. doi: 10.1111/j.1365-2958.1991.tb00799.x. [DOI] [PubMed] [Google Scholar]
  10. Coplin D. L., Rowan R. G., Chisholm D. A., Whitmoyer R. E. Characterization of plasmids in Erwinia stewartii. Appl Environ Microbiol. 1981 Oct;42(4):599–604. doi: 10.1128/aem.42.4.599-604.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dolph P. J., Majerczak D. R., Coplin D. L. Characterization of a gene cluster for exopolysaccharide biosynthesis and virulence in Erwinia stewartii. J Bacteriol. 1988 Feb;170(2):865–871. doi: 10.1128/jb.170.2.865-871.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Falkenstein H., Bellemann P., Walter S., Zeller W., Geider K. Identification of Erwinia amylovora, the Fireblight Pathogen, by Colony Hybridization with DNA from Plasmid pEA29. Appl Environ Microbiol. 1988 Nov;54(11):2798–2802. doi: 10.1128/aem.54.11.2798-2802.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gamborg O. L., Miller R. A., Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1968 Apr;50(1):151–158. doi: 10.1016/0014-4827(68)90403-5. [DOI] [PubMed] [Google Scholar]
  14. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  16. Houng H. S., Kopecko D. J., Baron L. S. Molecular cloning and physical and functional characterization of the Salmonella typhimurium and Salmonella typhi galactose utilization operons. J Bacteriol. 1990 Aug;172(8):4392–4398. doi: 10.1128/jb.172.8.4392-4398.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jiang X. M., Neal B., Santiago F., Lee S. J., Romana L. K., Reeves P. R. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. doi: 10.1111/j.1365-2958.1991.tb00741.x. [DOI] [PubMed] [Google Scholar]
  18. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  19. Leigh J. A., Lee C. C. Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules. J Bacteriol. 1988 Aug;170(8):3327–3332. doi: 10.1128/jb.170.8.3327-3332.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lemaire H. G., Müller-Hill B. Nucleotide sequences of the gal E gene and the gal T gene of E. coli. Nucleic Acids Res. 1986 Oct 10;14(19):7705–7711. doi: 10.1093/nar/14.19.7705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lesse A. J., Campagnari A. A., Bittner W. E., Apicella M. A. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Immunol Methods. 1990 Jan 24;126(1):109–117. doi: 10.1016/0022-1759(90)90018-q. [DOI] [PubMed] [Google Scholar]
  22. Majumdar A., Adhya S. Demonstration of two operator elements in gal: in vitro repressor binding studies. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6100–6104. doi: 10.1073/pnas.81.19.6100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marinus M. G., Carraway M., Frey A. Z., Brown L., Arraj J. A. Insertion mutations in the dam gene of Escherichia coli K-12. Mol Gen Genet. 1983;192(1-2):288–289. doi: 10.1007/BF00327681. [DOI] [PubMed] [Google Scholar]
  24. Maskell D. J., Szabo M. J., Butler P. D., Williams A. E., Moxon E. R. Molecular analysis of a complex locus from Haemophilus influenzae involved in phase-variable lipopolysaccharide biosynthesis. Mol Microbiol. 1991 May;5(5):1013–1022. doi: 10.1111/j.1365-2958.1991.tb01874.x. [DOI] [PubMed] [Google Scholar]
  25. Metzger M., Bellemann P., Schwartz T., Geider K. Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells. Nucleic Acids Res. 1992 May 11;20(9):2265–2270. doi: 10.1093/nar/20.9.2265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moreno F., Rodicio R., Herrero P. A new colorimetric assay for UDP-glucose 4-epimerase activity. Cell Mol Biol Incl Cyto Enzymol. 1981;27(6):589–592. [PubMed] [Google Scholar]
  27. Pech J. C., Romani R. J. Senescence of Pear Fruit Cells Cultured in a Continuously Renewed, Auxin-deprived Medium. Plant Physiol. 1979 Nov;64(5):814–817. doi: 10.1104/pp.64.5.814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Robertson B. D., Frosch M., van Putten J. P. The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide. Mol Microbiol. 1993 May;8(5):891–901. doi: 10.1111/j.1365-2958.1993.tb01635.x. [DOI] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sedmak J. J., Grossberg S. E. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem. 1977 May 1;79(1-2):544–552. doi: 10.1016/0003-2697(77)90428-6. [DOI] [PubMed] [Google Scholar]
  31. Simon R., Quandt J., Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989 Aug 1;80(1):161–169. doi: 10.1016/0378-1119(89)90262-x. [DOI] [PubMed] [Google Scholar]
  32. Spratt B. G., Hedge P. J., te Heesen S., Edelman A., Broome-Smith J. K. Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene. 1986;41(2-3):337–342. doi: 10.1016/0378-1119(86)90117-4. [DOI] [PubMed] [Google Scholar]
  33. Wei Z. M., Laby R. J., Zumoff C. H., Bauer D. W., He S. Y., Collmer A., Beer S. V. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science. 1992 Jul 3;257(5066):85–88. doi: 10.1126/science.1621099. [DOI] [PubMed] [Google Scholar]
  34. Yarmolinsky M. B., Wiesmeyer H., Kalckar H. M., Jordan E. HEREDITARY DEFECTS IN GALACTOSE METABOLISM IN ESCHERICHIA COLI MUTANTS, II. GALACTOSE-INDUCED SENSITIVITY. Proc Natl Acad Sci U S A. 1959 Dec;45(12):1786–1791. doi: 10.1073/pnas.45.12.1786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zimmermann S., Hahlbrock K. Light-induced changes of enzyme activities in parsley cell suspension cultures. Purification and some properties of phenylalanine ammonia-lyase (E.C.4.3.1.5). Arch Biochem Biophys. 1975 Jan;166(1):54–62. doi: 10.1016/0003-9861(75)90364-1. [DOI] [PubMed] [Google Scholar]

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