Mutation or Increased Copy Number of nodE Has No Effect on the Spectrum of Chitolipooligosaccharide Nod Factors Made By Rhizobiumleguminosarum bv. trifolii

Saleela Philip-Hollingsworth,Guy G Orgambide,James J Bradford,Damon K Smith,Rawle I Hollingsworth,Frank B Dazzo

doi:10.1074/jbc.270.36.20968

Saleela Philip-Hollingsworth, Guy G Orgambide + Show 4 more

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https://doi.org/10.1074/jbc.270.36.20968

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Abstract

The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determined unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont. We evaluated nodE function by examining CLOS structures made by wild-type R. leguminosarum bv. trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843. 1H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species. We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H. P., Bloemberg, G. V., van Brussel, A. A. N., Lugtenberg, B. J. J., van der Drift, K. M. G. M., Haverkamp, J., and Thomas-Oates, J. E. (1995) Mol. Plant-Microbe Interact. 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species. No evidence for nodE-dependent CLOSs was found for these strains. These results indicate a need to revise the current model to explain how nodE determines host range in the R. leguminosarum-legume symbiosis.

Highlights

Rhizobium, Bradyrhizobium, and Azorhizobium are bacterial genera that form N2-fixing nodules on legume roots
We have critically evaluated the proposed function of nodE by performing detailed structural analyses of CLOS species made by wild-type ANU843, an isogenic nodE::Tn5 mutant derivative ANU297, and a recombinant strain ANU845 pRtRF101 containing the cloned 14-kb HindIII pSym nod region of ANU843 on multiple copy plasmid pWB5a introduced into the pSym-cured derivative ANU845
Our experiments reveal that ANU843 makes a large diversity of major and minor CLOS species, which does not change with impairment of nodE function or increased nod copy number

Summary

Introduction

Bradyrhizobium, and Azorhizobium are bacterial genera that form N2-fixing nodules on legume roots In this symbiosis, the plant produces flavonoids that activate bacterial expression of nod genes necessary for production of “Nod factors” involved in infection and nodulation of the corresponding host plant [1,2,3,4]. We have critically evaluated the proposed function of nodE by performing detailed structural analyses of CLOS species made by wild-type ANU843, an isogenic nodE::Tn5 mutant derivative ANU297, and a recombinant strain ANU845 pRtRF101 containing the cloned 14-kb HindIII pSym nod region of ANU843 on multiple copy plasmid pWB5a introduced into the pSym-cured derivative ANU845. Our experiments reveal that ANU843 makes a large diversity of major and minor CLOS species, which does not change with impairment of nodE function or increased nod copy number This spectrum of CLOSs does not, include molecules containing tri- or tetraunsaturated fatty acids.

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