Dissecting the biosynthetic pathway and biological significance of unique lipopolysaccharide structures in bacterial nitrogen fixing endosymbionts
Brown, Dusty Barton
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Rhizobium are gram negative bacteria that form nitrogen fixing symbiosis (NFS) with compatible leguminous plants. Lipopolysaccharide (LPS) is a major cell surface molecule produced by gram negative bacteria and is crucial for establishing complete NFS in several symbiotic systems. In order to gain a more detailed understanding of LPS and its role in NFS, it is necessary to elucidate the structure, genes, and gene products. The general structure of LPS has been solved for the model organisms Rhizboium leguminosarum bv. viciae 3841 and R. etli bv. phaseolus CE3 and most of the biosynthetic genes have been discovered. LPS contains three domains; hydrophobic lipid A, core oligosaccharide, and O-chain polysaccharide. The O-chain is absolutely required to establish NFS, however, the function of the core and lipid A is unknown. The lipid A and core are highly galacturonosylated (GalA) containing one terminal 4` α-GalA on the lipid A and three terminal α-GalAs in the core. Inner membrane Rhizobium glycosyl transferases (RgtABCD) transfer GalA from the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA) to the lipid A and core. The lipid A portion contains the unique very long chain fatty acid (VLCFA, 27-hydroxyoctacosanoic) also found in intracellular mammalian pathogens. VLCFA is built off the special acyl carrier protein AcpXL by specific fatty acid biosynthetic elongating enzymes. R. etli produces 3-O-deacylated lipid A, attributed to outer membrane 3-O-deacylase activity also observed in mammalian pathogens due to the action of the outer membrane enzyme PagL. Interestingly, the endosymbiont Mesorhizobium loti and plant growth promoting bacteria Azospirillum lipoferum produce unusual lipid A α-1,1-GalA. In this work we describe the discovery of the lipid A 4` GalA transferase gene rgtD, the Dod-P-GalA biosynthetic gene rgtE, and the M. loti α-1,1-GalA transferase gene rgtF as well as the 3-O-deacylase gene pagL in R. etli. We have prepared single mutants in the rgtABCDE genes from R. leguminosarum 3841, the pagL gene from R. etli CE3, and the acpXL gene from R. leguminosarum bv. phaseolus 8002 and describe the effects of these mutations on LPS structure, synthesis, membrane stability, and symbiosis.