Molecular and General Genetics MGG

, Volume 213, Issue 1, pp 155–162 | Cite as

Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: Their primary structure and their interaction with psi and other nodulation genes

  • D. Borthakur
  • R. F. Barker
  • J. W. Latchford
  • L. Rossen
  • A. W. B. Johnston


Strains of Rhizobium leguminosarum (R. l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas. It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar. When peas were co-inoculated with pss mutant derivatives of a strain of R.l. bv viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix+ nodules were pressumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere). Bacteria from the Fix- nodules contained, exclusively, the strain lacking its sym plasmid. When pss mutant strains were co-inoculated with a Nod- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix+ nodules were formed, all of which were occupiced solely by the nodD mutant strain. Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development. Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability. Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi. The predicted polypeptide product of one of the pss genes had a hydrophobic aminoterminal region, suggesting that it may be located in the membrane. Since the psi gene product may also be associated with the bacterial membrane, the products of psi and pss may interact with each other.

Key words

DNA sequence Exopolysaccharide Nodulation psi pss Rhizobium leguminosarum 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bagdasarian M, Lurz R, Ruckert B, Franklin FCH, Bagdasarian MM, Frey J, Timmis KN (1981) Specific-purpose plasmid cloning vectors: II. Broad host-range high copy number, RSF1010-derived vectors and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247Google Scholar
  2. Barker RF, Idher KB, Thompson DV, Kemp JD (1983) Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi:15955. Plant Mol Biol 2:335–350Google Scholar
  3. Barriere GC, Barber CE, Daniels, MJ (1986) Molecular cloning of genes involved in the production of the extracellular polysaccharide xanthan by Xanthomonas campestris pv. campestris.Int J Biol Macromol 8:372–374Google Scholar
  4. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 83:188–198Google Scholar
  5. Beynon JL, Beringer JE, Johnston AWB (1980) Plasmids and host range in Rhizobium leguminosarum and Rhizobium phaseoli. J Gen Microbiol 120:421–429Google Scholar
  6. Bibb MJ, Findlay PR, Johnson MW (1984) The relationship between base composition and codon usage in bacterial genes and its use in the simple and reliable identification of protein-coding sequences. Gene 30:157–166Google Scholar
  7. Borthakur D, Johnston AWB (1987) Sequence of psi, a gene on the symbiotic plasmid of Rhizobium phaseoli which inhibits exopolysaccharide synthesis and nodulation and demonstration that its transcription is inhibited by psr, another gene on the symbiotic plasmid. Mol Gen Genet 207:149–154Google Scholar
  8. Borthakur D, Downie JA, Johnston AWB, Lamb JW (1985) psi, a plasmid-linked Rhizobium phaseoli gene which inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation. Mol Gen Genet 200:278–282Google Scholar
  9. Borthakur D, Barber CE, Lamb JW, Daniels MJ, Downie JA, Johnston AWB (1986) A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans of R. phaseoli and is corrected by cloned DNA from the phytopathogen Xanthomonas. Mol Gen Genet 203:320–323Google Scholar
  10. Borthakur D, Lamb JW, Johnston AWB (1987) Identification of two classes of Rhizobium phaseoli genes required for melanin synthesis, one of which is required for nitrogen fixation and activates the transcription of the other. Mol Gen Genet 207:155–160Google Scholar
  11. Chakrovorty AK, Zurkowski W., Shine J, Rolfe BG (1982) Symbiotic nitrogen fixation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and effective nodulation. J Mol Appl Genet 1:585–596Google Scholar
  12. Chen H, Rolfe BG (1987) Cooperativity between Rhizobium mutant strains: induction of nitrogen-fixing nodules on the tropical legume Leucaena leucocephala. J Plant Physiol 127:307–322Google Scholar
  13. Chen H, Batley M, Redmond J, Rolfe BG (1985) Alteration of the effective nodulation properties of a fast growing broad host range Rhizobium due to changes in exopolysaccharide synthesis. J Plant Physiol120:331–349Google Scholar
  14. Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang X-W, Finlay DR, Helinski DR (1985) Plasmids related to the broad host range vector pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13:149–153Google Scholar
  15. Djordjevic SP Chen H, Batley M, Redmond JW, Rolfe BG (1987) Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous polysaccharides. J Bacteriol 169:53–60Google Scholar
  16. Downie JA, Hombrecher G, Ma QS, Knight CD, Wells B, Johnston AWB (1983) Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity. Mol Gen Genet 190:359–365Google Scholar
  17. Downie JA, Knight CD, Johnston AWB, Rossen L (1985) Identification of genes and gene products involved in the nodulation of peas by Rhizobium leguminosarum. Mol Gen Genet 198:255–262Google Scholar
  18. Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652Google Scholar
  19. Firmin JL, Wilson KE, Rossen L, Johnston AWB (1986) Flavonoid induction of nodulation genes in Rhizobium reversed by other compounds present in plants. Nature 324:90–92Google Scholar
  20. Friedman AM, Long SR, Brown SE, Buikema WJ, Ausubel FM (1982) Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296Google Scholar
  21. Hirsch AM, Kuldau GA, Klein S, Signer ER (1985) Coinoculation with symbiotically defective mutants ofRhizobium meliloti. In: Evans HJ, Bottomley P(eds) Nitrogen fixation progress. Nijhoff, Dordrecht p 142Google Scholar
  22. Hirsch PR (1979) Plasmid-determined bacteriocin production in Rhizobium leguminosarum. J Gen Microbiol 112:135–142Google Scholar
  23. Hynes MF, Simon R, Niehaus K, Labes M, Puhler A (1986) The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfafa. Mol Gen Genet 202:356–362Google Scholar
  24. Johnston AWB, Beynon JL, Buchanan-Wollaston AV, Setchell SM, Hirsch PR, Beringer JE (1978) High frequency transfer of nodulation ability between strains and species of Rhizobium. Nature 276:634–636Google Scholar
  25. Johnston AWB, Hombrecher G, Brewin NJ, Cooper MC (1982) Two transmissible plasmids in Rhizobium leguminosarum strain 300. J Gen Microbiol 128:85–93Google Scholar
  26. Klein S, Hirsch AM, Smith CA, Signer ER (1987) Coinoculation with symbiotically defective mutants of Rhizobium meliloti. In: Verma DPS, Brisson N (eds) Molecular genetics of plant-microbe interactions. Nijhoff, Dordrecht pp 179–181Google Scholar
  27. Knight CD, Rossen L, Robertson JG, Wells B, Downie JA (1986) Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection. J Bacteriol 166:552–558Google Scholar
  28. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132Google Scholar
  29. Lamb JW, Hombrecher G, Johnston AWB (1982) Plasmid-determined nodulation and nitrogen fixation abilities in Rhizobium phaseoli. Mol Gen Genet 86:449–452Google Scholar
  30. Lamb JW, Downie JA, Johnston AWB (1985) Cloning of the nodulation (nod) genes of Rhizobium phaseoli and their homology to R. leguminosarum nod genes. Gene 4:235–241Google Scholar
  31. Leigh JA, Singer ER, Walker GC (1985) Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci USA 82:6231–6235Google Scholar
  32. Leigh JA, Reed JW, Hanks JF, Hirsch AM, Walker GW (1987) Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion. Cell 51:579–587Google Scholar
  33. Long SR (1984) Genetics of Rhizobium nodulation. In: Kosuge T, Nester EW (eds) Plant microbe interactions; vol 1, Molecular and genetic perspectivesGoogle Scholar
  34. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  35. Maxam A, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560Google Scholar
  36. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  37. Rolfe BG, Gresshoff PM, Shine J, Vincent JW (1980) Interaction between a non-nodulating and an ineffective mutant of Rhizobium trifolii resulting in effective (nitrogen fixing) nodules. Appl Environ Microbiol 39:449–452Google Scholar
  38. Rossen L, Shearman CA, Johnston AWB, Downie JA (1985) The nodD gene of Rhizobium leguminosarum is autoregulatory and in the presence of plant root exudate induces the nodABC genes. EMBO J 4:3369–3374Google Scholar
  39. Rossen L, Davis EO, Johnston AWB (1987) Plant-induced genes involved in host specificity and early stages of nodulation. TIBS 12:430–433Google Scholar
  40. Sanders R, Raleigh E, Signer E (1981) Lack of correlation between extracellular polysaccharide and nodulation ability in Rhizobium. Nature 292:148–149Google Scholar
  41. Scott KF (1986) Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp. (Parasponia). Nucleic Acids Res 14:2905–2919Google Scholar
  42. Shearman CA, Rossen L, Johnston AWB, Downie JA (1986) The Rhizobium leguminosarum nodulation gene nodF encodes a protein similar to acyl carrier protein and is regulated by nodF plus a factor in pea root exudate. EMBO J 5:647–652Google Scholar
  43. Stachel SE, An D, Flores C, Nester EW (1985) A Tn3 lacZ transposon for the random generation of β-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J 4:891–898Google Scholar
  44. Staden R (1984) Measurement of the effects that coding for a protein has on DNA sequence and their use for finding genes. Nucleic Acids Res 12:551–567Google Scholar
  45. Sutherland IW (1985) Biosynthesis and composition of Gram negative bacterial extracellular and wall polysaccharides. Annu Rev Microbiol 39:243–270Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • D. Borthakur
    • 1
  • R. F. Barker
    • 2
  • J. W. Latchford
    • 1
  • L. Rossen
    • 1
  • A. W. B. Johnston
    • 1
  1. 1.John Innes InstituteNorwichUK
  2. 2.Plant Breeding InstituteCambridgeUK

Personalised recommendations