The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determine 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 ANU843.1 H-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. leguminosarumlegume symbiosis.Rhizobium, Bradyrhizobium, and Azorhizobium are bacterial genera that form N 2 -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 (1-4). These Nod factors are chitolipooligosaccharides (CLOSs) 1 consisting of ␤-1,4-linked oligomers of N-acetylglucosamine bearing an amide-linked fatty acyl moiety at the nonreducing end and may contain other substituents (e.g. Oacetyl, sulfate, etc.) that make their biological activity hostspecific (5). The current model for nod functions is that the common nod genes encode enzymes that synthesize the common backbone of CLOSs, and the host-specific nod genes encode enzymes that introduce these modifications in CLOS structures making them host-specific (6, 7). Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) is the bacterial symbiont of the legume host, clover (Trifolium spp.). In the most thoroughly studied wild-type strain (ANU843), the ability to nodulate white clover is controlled by regulatory (nodD), common (nodABCIJ), and hostspecific (nodFERL, nodMN) nod genes residing within a 14-kb region on its resident symbiotic plasmid (pSym) (8, 9). Elegant studies have shown that NodE is the main determinant of nodulation host range for R. trifolii and its closest relative, the pea symbiont, R. leguminosarum bv. viciae (10, 11). Tn5 disruption of nodE (but not genes downstream of nodE) in ANU843 results in a unique dual phenotype, which is defective in nodula...

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