Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Aug;170(8):3327–3332. doi: 10.1128/jb.170.8.3327-3332.1988

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

J A Leigh 1, C C Lee 1
PMCID: PMC211298  PMID: 3403505

Abstract

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.

Full text

PDF
3327

Images in this article

3329Image
on p.3329

Selected References

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

  1. Cangelosi G. A., Hung L., Puvanesarajah V., Stacey G., Ozga D. A., Leigh J. A., Nester E. W. Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions. J Bacteriol. 1987 May;169(5):2086–2091. doi: 10.1128/jb.169.5.2086-2091.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carlson R. W., Kalembasa S., Turowski D., Pachori P., Noel K. D. Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development. J Bacteriol. 1987 Nov;169(11):4923–4928. doi: 10.1128/jb.169.11.4923-4928.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chakravorty A. K., Zurkowski W., Shine J., Rolfe B. G. Symbiotic nitrogen fixation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and effective nodulation. J Mol Appl Genet. 1982;1(6):585–596. [PubMed] [Google Scholar]
  4. Darveau R. P., Hancock R. E. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol. 1983 Aug;155(2):831–838. doi: 10.1128/jb.155.2.831-838.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Vos G. F., Walker G. C., Signer E. R. Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives. Mol Gen Genet. 1986 Sep;204(3):485–491. doi: 10.1007/BF00331029. [DOI] [PubMed] [Google Scholar]
  6. Djordjevic S. P., Chen H., Batley M., Redmond J. W., Rolfe B. G. Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides. J Bacteriol. 1987 Jan;169(1):53–60. doi: 10.1128/jb.169.1.53-60.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Douglas C. J., Halperin W., Nester E. W. Agrobacterium tumefaciens mutants affected in attachment to plant cells. J Bacteriol. 1982 Dec;152(3):1265–1275. doi: 10.1128/jb.152.3.1265-1275.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Douglas C. J., Staneloni R. J., Rubin R. A., Nester E. W. Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol. 1985 Mar;161(3):850–860. doi: 10.1128/jb.161.3.850-860.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dylan T., Ielpi L., Stanfield S., Kashyap L., Douglas C., Yanofsky M., Nester E., Helinski D. R., Ditta G. Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4403–4407. doi: 10.1073/pnas.83.12.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Finan T. M. Genetic and physical analyses of group E exo- mutants of Rhizobium meliloti. J Bacteriol. 1988 Jan;170(1):474–477. doi: 10.1128/jb.170.1.474-477.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Finan T. M., Hartweig E., LeMieux K., Bergman K., Walker G. C., Signer E. R. General transduction in Rhizobium meliloti. J Bacteriol. 1984 Jul;159(1):120–124. doi: 10.1128/jb.159.1.120-124.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Finan T. M., Hirsch A. M., Leigh J. A., Johansen E., Kuldau G. A., Deegan S., Walker G. C., Signer E. R. Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell. 1985 Apr;40(4):869–877. doi: 10.1016/0092-8674(85)90346-0. [DOI] [PubMed] [Google Scholar]
  13. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986 Jul;167(1):66–72. doi: 10.1128/jb.167.1.66-72.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geremia R. A., Cavaignac S., Zorreguieta A., Toro N., Olivares J., Ugalde R. A. A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharide but fails to form beta-(1----2) glucan. J Bacteriol. 1987 Feb;169(2):880–884. doi: 10.1128/jb.169.2.880-884.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harada T., Amemura A., Jansson P. E., Lindberg B. Comparative studies of polysaccharides elaborated by Rhizobium, Alceligenes, and Agrobacterium. Carbohydr Res. 1979 Dec;77:285–288. doi: 10.1016/s0008-6215(00)83821-5. [DOI] [PubMed] [Google Scholar]
  16. Jansson P. E., Kenne L., Lindberg B., Ljunggren H., Lönngren J., Rudén U., Svensson S. Demonstration of an octasaccharide repeating unit in the extracellular polysaccharide of Rhizobium meliloti by sequential degradation. J Am Chem Soc. 1977 May 25;99(11):3812–3815. doi: 10.1021/ja00453a049. [DOI] [PubMed] [Google Scholar]
  17. Leigh J. A., Reed J. W., Hanks J. F., Hirsch A. M., Walker G. C. Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion. Cell. 1987 Nov 20;51(4):579–587. doi: 10.1016/0092-8674(87)90127-9. [DOI] [PubMed] [Google Scholar]
  18. Leigh J. A., Signer E. R., Walker G. C. Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6231–6235. doi: 10.1073/pnas.82.18.6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levery S. B., Hakomori S. Microscale methylation analysis of glycolipids using capillary gas chromatography-chemical ionization mass fragmentography with selected ion monitoring. Methods Enzymol. 1987;138:13–25. doi: 10.1016/0076-6879(87)38004-8. [DOI] [PubMed] [Google Scholar]
  20. Marks J. R., Lynch T. J., Karlinsey J. E., Thomashow M. F. Agrobacterium tumefaciens virulence locus pscA is related to the Rhizobium meliloti exoC locus. J Bacteriol. 1987 Dec;169(12):5835–5837. doi: 10.1128/jb.169.12.5835-5837.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miller K. J., Reinhold V. N., Weissborn A. C., Kennedy E. P. Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues. Biochim Biophys Acta. 1987 Jul 10;901(1):112–118. doi: 10.1016/0005-2736(87)90262-8. [DOI] [PubMed] [Google Scholar]
  22. Noel K. D., Vandenbosch K. A., Kulpaca B. Mutations in Rhizobium phaseoli that lead to arrested development of infection threads. J Bacteriol. 1986 Dec;168(3):1392–1401. doi: 10.1128/jb.168.3.1392-1401.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Puvanesarajah V., Schell F. M., Stacey G., Douglas C. J., Nester E. W. Role for 2-linked-beta-D-glucan in the virulence of Agrobacterium tumefaciens. J Bacteriol. 1985 Oct;164(1):102–106. doi: 10.1128/jb.164.1.102-106.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Szu S. C., Zon G., Schneerson R., Robbins J. B. Ultrasonic irradiation of bacterial polysaccharides. Characterization of the depolymerized products and some applications of the process. Carbohydr Res. 1986 Sep 1;152:7–20. doi: 10.1016/s0008-6215(00)90283-0. [DOI] [PubMed] [Google Scholar]
  25. Thomashow M. F., Karlinsey J. E., Marks J. R., Hurlbert R. E. Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment. J Bacteriol. 1987 Jul;169(7):3209–3216. doi: 10.1128/jb.169.7.3209-3216.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tolmasky M. E., Staneloni R. J., Leloir L. F. Lipid-bound saccharides in Rhizobium meliloti. J Biol Chem. 1982 Jun 25;257(12):6751–6757. [PubMed] [Google Scholar]
  27. Tolmasky M. E., Staneloni R. J., Ugalde R. A., Leloir L. F. Lipid-bound sugars in Rhizobium meliloti. Arch Biochem Biophys. 1980 Aug;203(1):358–364. doi: 10.1016/0003-9861(80)90187-3. [DOI] [PubMed] [Google Scholar]
  28. Toro N., Olivares J. Analysis of Rhizobium meliloti Sym Mutants Obtained by Heat Treatment. Appl Environ Microbiol. 1986 May;51(5):1148–1150. doi: 10.1128/aem.51.5.1148-1150.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Watanabe H., Igari D., Tanahashi Y., Harada K., Saito M. Measurements of size and weight of prostate by means of transrectal ultrasonotomography. Tohoku J Exp Med. 1974 Nov;114(3):277–285. doi: 10.1620/tjem.114.277. [DOI] [PubMed] [Google Scholar]
  30. York W. S., McNeil M., Darvill A. G., Albersheim P. Beta-2-linked glucans secreted by fast-growing species of Rhizobium. J Bacteriol. 1980 Apr;142(1):243–248. doi: 10.1128/jb.142.1.243-248.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zorreguieta A., Ugalde R. A. Formation in Rhizobium and Agrobacterium spp. of a 235-kilodalton protein intermediate in beta-D(1-2) glucan synthesis. J Bacteriol. 1986 Sep;167(3):947–951. doi: 10.1128/jb.167.3.947-951.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES