The lipopolysaccharide galacturonic acid modification pathway in Rhizobium

The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in host interactions and outer membrane stability. The lipid A and core regions of LPS in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In particular, the R. leguminosarum LPS inner core is modified with several galacturonic acid (GalA) moieties.; We have identified a hybrid cosmid (pSGAT), containing a 22 kb R. leguminosarum 3841 genomic DNA insert that directs the overexpression of three GalA transferases. Two of these enzymes modify radiolabeled LPS precursor substrates on the distal Kdo, as indicated by mild acid hydrolysis. Sequencing of a 7 kb DNA fragment derived from pSGAT reveals the presence of three putative membrane-bound glycosyltransferases (now designated RgtA, RgtB and RgtC). Transfer via tri-parental mating of the 7 kb fragment into Sinorhizobium meliloti 1021, a strain that lacks these inner core GalA modifications, resulted in the heterologous expression of GalA transferase activity.; Reconstitution experiments with the individual genes demonstrate that the activity of RgtA precedes, and is necessary for, the subsequent activity of RgtB. We also demonstrate RgtC to be the core mannose GalA transferase that acts after RgtA and RgtB. Electrospray ionization/tandem mass spectrometry (ESI/MS) and gas-liquid chromatography (GC/MS) techniques have been used to analyze the product generated in vitro by RgtA. The analyses confirm the presence of GalA.; Differences in RgtA activity upon heterologous expression in Sinorhizhobium meliloti 1021 versus E. coli appeared to reflect the presence or absence of a donor substrate. RgtA activity in E. coli could be reconstituted upon addition of Rhizobiaceae membranes or total lipids. Consequently, we have identified dodecaprenyl (C60) phosphate-GalA as a minor novel lipid in R. leguminosarum 3841. The lipid has been purified using mild base hydrolysis and DEAE-cellulose column chromatography. The structure of the GalA donor lipid has been characterized by ESI/MS and GC/MS methods. The purified lipid supports the in vitro transfer of GalA to the model substrate, using membranes of E. coli cells expressing RgtA. This work represents the first definitive characterization of a lipid-linked GalA donor and its allied enzymes.