The Function of the Rhizobial NodABC and NodFEL Operons in the Biosynthesis of Lipo-Oligosaccharides

Abstract

In a Sym plasmid-cured strain of R.leguminosarum the presence of the NodA,B,C,F,E,L proteins is sufficient for production and secretion of the wild type lipo-oligosaccharides. Using PCR technology we have constructed plasmids of various incompatibility groups (IncW, IncP, IncQ) which contain the separated single genes, under control of a common promoter (nodA- or T7-derived) and a synthetic ribosomal binding site. Rhizobium strains harbouring various combinations of the single nod gene-containing plasmids were tested for the production of lipooligosaccharides and the results show that: (i) The absence of either of the NodA, NodB or NodC proteins prevents the production of lipo-oligosaccharide molecules, (ii) absence of NodF or NodE proteins prevents the production of lipooligosaccharides which contain a highly unsaturated (C18:4) fatty acyl moiety, (iii) absence of the NodL protein prevents the addition of the O-acetyl group on the non-reducing terminal sugar. The biochemical function of these Nod proteins is further studied by the analysis of biosynthetic intermediates of the lipooligosaccharides in strains producing the various combinations of Nod proteins. Furthermore, we have set up assays for in vitro biochemical activity of the Nod proteins at present focusing on NodA, NodB, NodC and NodL. The obtained results suggest that NodC functions as an N-acetylglucosaminyltransferase involved in the synthesis of the sugar backbone whereas NodA and NodB are involved in the de-N-acetylation and/or acylation of the non-reducing terminal Nacetylglucosamine. The NodL protein is an O-acetyl transferase which can use non-O-acetylated lipo-oligosaccharides or chitin oligomers as substrates.