Archives of Microbiology

, Volume 155, Issue 1, pp 35–41 | Cite as

Synthesis of β(1–2)glucan in Rhizobium loti

Expression of Agrobacterium tumefaciens chvB virulence region
  • Viviana Lepek
  • Yolanda Navarro de Navarro
  • Rodolfo A. Ugalde
Original Papers


Fast growing strains of Rhizobium loti isolated from nodules of Lotus tenuis of the flooding Pampas of Argentina produced cellular β(1–2)glucans having a higher degree of polymerization and more anionic substituents than β(1–2)glucans accumulated by Agrobacterium tumefaciens cells. Inner membranes of R. loti contained a 235 kDa β(1–2)glucan intermediate protein indistinguishable by polyacrylamide gel electrophoresis from the intermediate protein present in A. tumefaciens inner membranes. Incubation of inner membrannes of R. loti with UDP-Gle led to the formation of neutral β(1–2)glucans with a higher degree of polymerization than glucans formed by A. tumefaciens inner membranes.

Introduction in R. loti strains of plasmid pCD523 containing A. tumefaciens chvA and chvB virulence regions yielded strains that accumulated 4 times more cellular β(1–2)glucans than wild type cells. This glucan was, regarding anionic substitution and degree of polymerization, indistinguishable from A. tumefaciens β(1–2)glucans. Furthermore inner membranes of these R. loti exoconjugant cells contained higher levels of the 235 kDa β(1–2)glucan intermediate protein and formed in vitro 8 times more neutral β(1–2)glucan with a degree of polymerization corresponding to A. tumefaciens β(1–2)glucan than inner membranes isolated from wild type cells.

It was concluded that A. tumefaciens chvB gene is expressed in R. loti and determined the degree of polymerization of β(1–2)glucan.

Key words

Rhizobium loti β(1–2)glucan Agrobacterium tumefaciens virulence region Lotus tenuis 



effective nodulation






trimethoprim resistence


tetracycline resistence


trichloroacetic acid


phenyl methyl sulfonyl fluoride


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  1. Altabe S, Iñon de Iannino N, de Mendoza D, Ugalde R (1990) Expression of the Agrobacterium tumefaciens chvB virulence region in Azospirillum spp J Bacteriol 172:2573–2567Google Scholar
  2. Banfalvi Z, Sakanyan B, Koncz C, Kiss A, Dusha I, Kondorosi A (1981) Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Mol Gen Genet 184:318–325Google Scholar
  3. Carlson RW, Shatters R, Duh JL, Turnbull E, Hanley B, Rolfe BG, Djordjewic A (1987) The isolation and partial characterization of the lipopolysaccharides from several Rhizobium trifolii mutants affected in root hair infection. Plant Physiol 84:421–427Google Scholar
  4. Chua K, Pankhurst CE, Macdonald PE, Hopcroft DH, Jarvis BDW, Scott DB (1985) Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti. J Bacteriol 162:335–343Google Scholar
  5. Dazzo FB, Truchet GL, Hrabak EM (1984) Specific enhancement of clover root hair infections by trifoli in a binding lipolopysaccharide from Rhizobium trifolii. Adv Nitrogen Fixat Res Proceeding of the 5th International Symposium on Nitrogen Fixation, The Netherlands 1983. 413Google Scholar
  6. Dische Z (1962) General color reactions. Methods Carbohydr Chem 1:478–492Google Scholar
  7. Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium melilott. Proc Natl Acad Sci USA 77:7347–7351Google Scholar
  8. Douglas CJ, Staneloni RJ, Rubin RA, Nester EW (1985) Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol 161:850–860Google Scholar
  9. Dylan CJ, Ielpi L, Stanfield S, Kashyap L, Douglas C, Yanofsky M, Nester EW, Helinski DR, Ditta G (1986) Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens. Proc Natl Acad Sci USA 83:4403–4407Google Scholar
  10. Finan TM, Hirsch AM, Leigh JA, Johansen EJ, Kuldau GA, Deegan S, Walker GC, Signer ER (1985) Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell 40:869–877Google Scholar
  11. Garfinkel DJ, Nester EW (1980) Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol 144:732–743Google Scholar
  12. Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW (1981) Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153Google Scholar
  13. Geremía RA, Cavaignac S, Zorreguieta A, Toro N, Olivares J, Ugalde RA (1987) A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharides but fails to form β(1–2)glucan. J Bacteriol 169:880–884Google Scholar
  14. Hombrecher G, Brewin NJ, Johnston AWB (1981) Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and Rhizobium phaseoli. Mol Gen Genet 182:133–136Google Scholar
  15. Hooykaas PJJ, van Brussel AAN, den Dalk-Ras H, van Slogteren GMS, Schilperoort RA (1981) Sym plasmid of Rhizobium trifolli expressed in different rhizobial species and Agrobacterium tumefaciens. Nature 291:351–353Google Scholar
  16. Iñon de Iannino N, Ugalde RA (1989) Biochemical characterization of avirulent Agrobacterium tumefaciens chvA mutants: synthesis and excretion of β-(1–2)glucan. J Bacteriol 171:2842–2849Google Scholar
  17. Jensen HL (1942) Nitrogen fixation in leguminous plants. I. General characters of root nodule bacteria isolated from species of Medicago and Trifollum. Proc Linn Soc NSW 66:98–108Google Scholar
  18. Jones WT, Macdonald PE, Jones SD, Pankhurst CE (1987) Peptidoglycan-bound polysaccharide associated with resistance of Rhizobium-loti strain NZP2037 to Lotus pedunculatus root flavolan. J Gen Microbiol 133:2617–2630Google Scholar
  19. Koizumi K, Okada Y, Horiyama S, Utamura T, Hisamatsu M, Amemura A (1983) Separation of cyclic (1–2) β-d-glucans (cyclosophoraoses) produced by Agrobacterium and Rhizobium and determination of their degree of polymerization by high performance liquid chromatography. J Chromatogr 265:89–96Google Scholar
  20. Leigh J, Signer ER, Walker GC (1985) Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci USA 82:6231–6235Google Scholar
  21. Leong S, Ditta GS, Helinski DR (1982) Heme biosynthesis in Rhizobium. Identification of a cloned gene coding for σ-aminolevulinic acid synthetase from Rhizobium meliloti. J Biol Chem 257:8724–8730Google Scholar
  22. McIntire FC, Peterson WH, Riker AJ (1942) A polysaccharide produced by the crown gall organism. J Biol Chem 143:491–496Google Scholar
  23. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  24. Miller KJ, Kennedy EP, Reinhold VN (1986) Osmotic adaptation by Gram-negative bacteria: possible role for periplasmic oligosaccharides. Science 231:48–51Google Scholar
  25. Osborn MJ, Munson R (1984) Separation of inner (cytoplasmic) and outer membranes of gram negative bacteria. Methods Enzymol 31A:642–653Google Scholar
  26. Pankhurst CE, Jones WT (1979) Effectiveness of Lotus root nodules. II. Relation between root nodule effectiveness and “in vitro” sensitivity of fast growing Lotus rhizobia to flavolans. J Exp Bot 30:1095–1107Google Scholar
  27. Pankhurst CE, Craig AS, Jones WT (1979) Effectiveness of Lotus root nodules. I. Morphology and flavolan content of nodules formed on Lotus pedunculatus by fast-growing Lotus rhizobia. J Exp Bot 30:1085–1093Google Scholar
  28. Pankhurst CE, Broughton WJ, Wieneke U (1983) Transfer of an indigenous plasmid of Rhizobium loti to other Rhizobia and Agrobacterium tumefaciens. J Gen Microbiol 129:2535–2543Google Scholar
  29. Puvanesarajah V, Schell FM, Stacey G, Douglas CJ, Nester EW (1985) Role for 2-linked-β-d-glucan in the virulence of Agrobacterium tumefaciens. J Bacteriol 164:102–106Google Scholar
  30. Stanfield SW, Ielpi L, O'Brochta D, Helinski DR, Ditta GS (1988) The ndvA gene product in Rhizobium meliloti is required for β-(1–2)glucan production and has homology to the ATP-binding export protein HlyB. J Bacteriol 170:3523–3530Google Scholar
  31. Ugalde RA, Coira JA, Brill WJ (1986a) Biosynthesis of a galactose and galacturonic acid-containing polysaccharide in Rhizobium meliloti. J Bacteriol 168:270–275Google Scholar
  32. Ugalde RA, Handelsman J, Brill WJ (1986b) Role of galactosyltransferase activity in phage sensitivity and nodulation competitiveness of Rhizobium meliloti. J Bacteriol 166:148–154Google Scholar
  33. Vincent M (1970) A manual for practical study of root nodule bacteria, Blackwell, OxfordGoogle Scholar
  34. Ward LJH, Rockman ES, Ball P, Jarvis BDW, Scott DB (1989) Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus. Mol Plant-Microbe Interact 2:224–232Google Scholar
  35. Wright A, Robbins PW (1965) The enzymatic synthesis of uridine diphospho[14C]glucose. Biochim Biophys Acta 104:594–596Google Scholar
  36. York WS, McNeil SM, Darwill AG, Albersheim P (1980) Beta-2-linked glucans secreted by the fast-growing species of Rhizobium. J Bacteriol 142:243–248Google Scholar
  37. Zorreguieta A, Geremía RA, Cavaignac S, Cangelosi GA, Nester EW, Ugalde RA (1988) Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene. Mol Plant Microb Interact 1:121–127Google Scholar
  38. Zorreguieta A, Tolmasky ME, Staneloni RJ (1985a) The enzymatic synthesis of β (1–2) glucans. Arch Biochem Biophys 238:368–372Google Scholar
  39. Zorreguieta A, Ugalde RA, Leloir LF (1985b) An intermediate in cyclic β (1–2) glucan biosynthesis. Biochem Biophys Res Commun 126:352–357Google Scholar
  40. Zorreguieta A, Ugalde RA (1986) Formation in Rhizobium and Agrobacterium spp. of a 235-kilodalton protein intermediate in β-d(1–2)glucan synthesis. J Bacteriol 167:947–951Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Viviana Lepek
    • 1
  • Yolanda Navarro de Navarro
    • 1
  • Rodolfo A. Ugalde
    • 1
  1. 1.Instituto de Investigaciones Bioquímicas “Fundación Campomar”CONICETBuenos AiresArgentina

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