LIPOPOLYSACCHARIDE CORE GLYCOSYLATION IN RHIZOBIUM-LEGUMINOSARUM - ANUNUSUAL MANNOSYL TRANSFERASE RESEMBLING THE HEPTOSYL TRANSFERASE-I OFESCHERICHIA-COLI

The lipopolysaccharide structure of the nitrogen-fixing bacterium Rhiz obium leguminosarum differs from that of Escherichia coli in several w ays, one of which is the sugar composition of the core. The E. coli in ner core consists of 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glyc ero-D-manno-heptose (heptose), while the inner core of R. leguminosaru m contains 2-keto-3-deoxy-D-manno-octulosonic acid (Hdo), mannose, gal actose, and galacturonic acid. The two Kdo residues and their Linkages appear to be identical in both species. The Linkages of heptose in E. coli and of mannose in R. leguminosarum to Kdo are both alpha 1-5. We now characterize a membrane-associated glycosyl transferase in R. leg uminosarum extracts that incorporates mannose into nascent lipopolysac charide, using Kdo(2)-lipid IVA as the acceptor and GDP-mannose (or sy nthetic ADP-mannose) as the donor. The mannosyl transferase is associa ted with the inner membrane. The apparent K-m values for GDP-mannose a nd Kdo(2)-lipid IVA are 4.3 mu M and 7.1 mu M, respectively, in the pr esence of excess co-substrate. Extracts of E. coli do not catalyze GDP -mannose-dependent glycosylation of Kdo(2)-lipid IVA, but they are act ive when ADP-mannose is substituted for GDP-mannose. Given the structu ral similarity of ADP-mannose to ADP-heptose, we examined the possibil ity that heptosyl transferase I of E. coli (the product of the rfaC ge ne) catalyzes mannose transfer from ADP-mannose to Kdo(2)-lipid IVA. E xtracts of E. coli mutants defective in the rfaC gene are unable carry out ADP-mannose-dependent glycosylation of Kdo(2)-lipid IVA. Plasmids bearing rfaC(+) not only restore the missing activity but also direct its overexpression. Our assay using ADP-mannose as a substitute for A DP-heptose (which is not readily available) should facilitate the puri fication and characterization of heptosyl transferase I of E. coli. Th e GDP-mannose-dependent enzyme of R. leguminosarum may represent a fun ctional equivalent of E. coli RfaC.