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 coli 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. coli 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. coli 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.