LIPOPOLYSACCHARIDE BIOSYNTHESIS IN RHIZOBIUM-LEGUMINOSARUM - NOVEL ENZYMES THAT PROCESS PRECURSORS CONTAINING 3-DEOXY-D-MANNO-OCTULOSONIC ACID

The lipopolysaccharide of Rhizobium leguminosarum differs from that of other Gram-negative organisms. R. leguminosarum lipid A lacks phospha te groups, but it contains a galacturonic acid residue at the 4'-posit ion and an aminogluconate moiety in place of the usual glucosamine 1-p hosphate unit. R. leguminosarum lipid A is esterified with a peculiar long chain fatty acid, 27-hydroxyoctacosanoate, not found in enteric G ramnegative bacteria, and the inner core of R. leguminosarum contains mannose and galactose in place of heptose. Despite these differences, the biosynthesis of R. leguminosarum lipid A is initiated by the same seven enzyme pathway as in Escherichia coli (Raetz, C. R. H. (1993) J. Bacteriol. 175, 5745-5753) to form the phosphorylated precursor, (Kdo )(2)-lipid IVA which is then processed differently. We now describe se veral novel Rhizobium-specific enzymes that recognize and modify (Kdo) (2)-lipid IVA. The 1- and 4'-phosphatases were detected using (Kdo)(2) -[1-P-32]-lipid IVA and (Kdo)(2)-[4'-P-32]-lipid IVA, respectively, as shown by release of P-32(i). In the presence of GDP-mannose and/or UD P-galactose, membranes of R. leguminosarum first transferred mannose a nd then galactose to (Kdo)(2)-[4'-P-32]-lipid IVA. In addition, at lea st two hydrophobic metabolites were generated from (Kdo)(2)-[4'-P-32]- lipid IVA in a manner that was dependent upon both membranes and a cyt osolic factor from R. leguminosarum. These compounds are attributed to novel acylations of (Kdo)(2)-[4'-P-32]-lipid IVA. E. coli membranes a nd cytosol did not catalyze any of the unique reactions detected in R. leguminosarum extracts. Our findings establish the conservation and v ersatility of (Hdo)(2)-lipid IVA as a lipid A precursor in bacteria.