STRUCTURAL IDENTIFICATION OF METABOLITES PRODUCED BY THE NODB AND NODC PROTEINS OF RHIZOBIUM-LEGUMINOSARUM

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosaccharides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express t he single nodA, nodB and nodC genes or combinations of the three. In v ivo radioactive labelling experiments, in which D[1-C-14]-glucosamine was used as a precursor, followed by mass spectrometric analysis of th e purified radiolabelled metabolic products, showed that Rhizobium str ains that only express the combination of the nodB and nodC genes do n ot produce lipo-chitin oligosaccharides but instead produce chitin oli gomers (mainly pentamers) which are devoid of the N-acetyl group on th e non-reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expr essed in addition to the nodBC genes the majority of metabolites conta in an additional O-acetyl substituent on the non-reducing terminal sug ar residue (designated NodBCL metabolites). The NodBC and NodBCL metab olites purified after in vivo labelling were compared with the radiola belled metabolites produced in vitro by Rhizobium bacterial cell lysat es to which UDP-N-acetyl-D-[U-C-14]-glucosamine was added using thin-l ayer chromatography. The results show that the lysates of strains whic h expressed the nodBC or nodBCL genes can also produce NodBC and NodBC L metabolites. The same results were obtained when the Nods and NodC p roteins were produced separately in two different strains. On the basi s of these and other recent results, we propose that NodB is a chitin oligosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase a nd, by default, NodA is involved in lipid attachment.