Nod Factor Requirements for Efficient Stem and Root Nodulation of the Tropical Legume Sesbania rostrata

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Carbamoylation of azorhizobial nod factors is mediated by NodU

Lipochitooligosaccharides (LCOs) synthesized by Azorhizobium caulinodans ORS571 are substituted at the nonreducing-terminal residue with a 6-O-carbamoyl group, LCO biosynthesis in A. caulinodans is dependent on the nodABCSUIJZnoeC operon, Until now, the role of the nodulation protein NodU in the synthesis of azorhizobial LCOs remained unclear, Based on sequence similarities and structural analysis of LCOs produced by a nodU mutant, a complemented nodU mutant, and Escherichia coli DH5 alpha expressing the nodABCSU genes, NodU was shown to be involved in the carbamoylation step

Biosynthesis of Azorhizobium caulinodans Nod factors: study of the activity of the NodABCS proteins by expression of the genes in Escherichia coli

By in vitro and in vivo studies with Escherichia coil expressing different combinations of the nodABCS genes of Azorhizobium caulinodans, Nod factor intermediates were identified and their structures determined using mass spectrometry. Substrate-product relationships were studied by time course experiments, and the Nod factor biosynthetic pathway was partially resolved. E. coil strains, harboring nodA and/or nocB, did not produce Nod metabolites, whereas the strain expressing nodC produced chitooligosaccharides. Thus, the first committed step was the production of the carbohydrate backbone. Bacitracin and tunicamycin did not affect this step, suggesting that undecaprenyl pyrophosphate-linked intermediates were not involved. The second step was the deacetylation of chitooligosaccharides by NodB since the E. coil strain expressing nodBC produced chitooligosaccharides, deacetylated at the non-reducing end and since the NodC products were precursors of the NodBC products. A strain expressing nodBCS produced N-methylated oligosaccharides, whereas a strain expressing nodCS produced unmethylated oligosaccharides. Time course experiments showed that methylation occurred after deacetylation. Thus, NodS acted after NodB. The NodBCS metabolites were partially converted to Lipo-chitooligosaccharides when the nodABCS genes were expressed, showing that NodA was involved in the acylation and acted after NodS

Nod factors of Azorhizobium caulinodans strain ORS571 can be glycosylated with an arabinosyl group, a fucosyl group, or both

In addition to the previously described arabinosylated Nod factors, Azorhizobium caulinodans can also produce fucosylated Nod factors and Nod factors that are both arabinosylated and fucosylated. The presence of a plasmid carrying extra copies of a subset of nod genes as well as bacterial growth conditions influence the relative proportion of carbamoylated, fucosylated, and arabinosylated Nod factors, By using a root hair formation assay, we demonstrate that the Nod factor glycosylations are important for biological activity on Sesbania rostrata roots