BIOSYNTHESIS OF A STRUCTURALLY NOVEL LIPID-A IN RHIZOBIUM-LEGUMINOSARUM - IDENTIFICATION AND CHARACTERIZATION OF 6 METABOLIC STEPS LEADING FROM UDP-GLCNAC TO 3-DEOXY-D-MANNO-2-OCTULOSONIC ACID(2) LIPID-IV(A)

Lipopolysaccharides (LPSs) are prominent structural components of the outer membranes of gram-negative bacteria. In Rhizobium spp. LPS funct ions as a determinant of the nitrogen-fixing symbiosis with legumes. L PS is anchored to the outer surface of the outer membrane by the lipid A moiety, the principal lipid component of the outer bacterial surfac e. Several notable structural differences exist between the lipid A of Escherichia coli and that of Rhizobium leguminosarum, suggesting that diverse biosynthetic pathways may also exist. These differences inclu de the lack of phosphate groups and the presence of a 4'-linked GalA r esidue in the fatter. However, we now show that UDP-GlcNAc plays a key role in the biosynthesis of lipid A in R. leguminosarum, as it does i n E. coli. P-32-labeled monosaccharide and disaccharide lipid A interm ediates from E. coli were isolated and tested as substrates in cell ex tracts of R. leguminosarum biovars phaseoli and viciae. Six enzymes th at catalyze the early steps of E. coli lipid A biosynthesis were also present in extracts of R. leguminosarum. Our results show that all the enzymes of the pathway leading to the formation of the intermediate 3 -deoxy-D-manno-2-octulosonic acid (Kdo(2)) lipid IVA are functional in both R. leguminosarum biovars. These enzymes include (i) UDP-GlcNAc 3 -O-acyltransferase; (ii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacety lase; (iii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcN N-acyltransferase; (iv ) disaccharide synthase; (v) 4'-kinase; and (vi) Kdo transferase. Our data suggest that the early steps in lipid A biosynthesis are conserve d and that the divergence leading to rhizobial lipid A may occur at a later stage in the pathway, presumably after the attachment of the Kdo residues.