Host Specificity of Rhizobium leguminosarum Is Determined by the Hydrophobicity of Highly Unsaturated Fatty Acyl Moieties of the Nodulation Factors. Herman P Spaink . Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. Guido V. Bloemberg(1) Anton A. N. van Brussel(1) Ben J. J. Lugtenberg(1), Koen M. G. M. van der Drift(2), Johan Haverkamp(2), and Jane E.Thomas-Oates(2). (1)Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands; (2)Bijvoet Center for Biomolecular Research, Department of Mass Spectrometry, Utrecht University, P.O.Box 80083, 3508 TB Utrecht, The Netherlands. MPMI 8:155-164. Accepted 31 October 1994 Accepted 20 October 1994. Copyright 1995 The American Phytopathological Society.

The nodE genes of Rhizobium leguminosarum bvs. trifolii and viciae strains are the major determinants of the host range of nodulation. Using mass spectrometry we have analyzed the structures of the major and minor lipo-oligosaccharides produced by two R. leguminosarum strains that differ only in the origin of the nod genes. The strain containing the nod genes of R. I. bv. viciae produces lipo-chitin oligosaccharide (LCO) molecules that contain the common cis-vaccenyl (C18:l) or a highly unsaturated acyl (C18:4) group that is determined by nodE (Spaink et al. Nature 354:125-130, 1991). Here we show that, in addition, minor quantities of analogous molecules that contain different common fatty acyl groups such as stearyl (C18:0), palmityl (C16:0), and palmiloyl (C16:l) groups are produced. The set of LCOs produced by the strain containing the R. I. bv. trifolii nod genes is similar, but major differences are found in the length and unsaturation of the fatty acyl groups of various LCOs. The R. I. bv. trifolii strain does not produce LCO molecules that contain the C18:4 fatty acyl group. Instead, a complex mixture of other unsaturated fatty acyl groups containing either two, three, or four double bonds is produced. These novel fatty acyl groups are more hydrophobic than the C18:4 fatty acyl group found in bv. viciae, as judged by high-performance liquid chromatography retention times, probably as a result of the longer carbon chain length, the lack of a cis double bond, or both. Another difference is that the relative amount of highly unsaturated fatty acid-containing LCOs produced by R. 1. bv. trifolii is much smaller. An isogenic R. I. bv. trifolii strain containing a Tn5 insertion in the nodE gene only produces LCOs containing the common fatty acyl groups and not the highly unsaturated types. The results indicate that the difference in the host range of the R. I. bvs. viciae and trifolii is determined by the overall hydrophobicity of the highly unsaturated fatty acyl moieties of LCOs rather than by a specific structural feature .