An additional GalAT, RgtE, is a bacterial like dolichol-phosphate mannosyl transferase (Dpm1) of the GT family 2 (GT-2) and is responsible for the biosynthesis of the Dod-P-GalA lipid donor. They belong to the glycosyl transferase (GT) family 39 (GT-39) and require the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA) for GalA transfer to the LPS.
The Rhizobium LPS core GalA transferases (GalATs Rgt A, B, and C), as well as the lipid A specific RgtD (1,4′)-GalAT, are 4-amino-4-deoxy-arabinosyl transferase (ArnT)-like ( Trent et al. 2012) and similar genes are present in the sequenced genomes of R. leguminosarum 3841 ( Kanjilal-Kolar et al. The genes encoding the glycosyl transferases responsible for GalA attachment to the lipid A and core have been determined in R. leguminosarum produce α-(1,4′) mono-galacturonosylated lipid A as well as three terminally linked GalAs on the LPS core. The nitrogen-fixing endosymbionts Rhizobium etli and R. aeolicus, the DAG disaccharide is bis-galacturonosylated with GalA attached to the lipid A 4′ (distal) and 1 (proximal) positions. 2008) and Aquifex aeolicus ( Plotz et al. Organisms that produce lipid A substituted with α-(1,1) GalA include Azospirillum lipoferum ( Choma and Komaniecka 2008), Caulobacter crescentus ( Smit et al.
The presence of phosphate, DAG and GalA has also been reported in the lipid A from the closely related strain Rhizobium loti NZP2213 ( Russa et al. The backbone comprises a β-(1,6)-2,3-diamino-2,3-dideoxyglucosamine (DAG) disaccharide that is substituted with phosphate at the 4′ position and contains an unusual GalA residue α-(1,1)- linked to the proximal DAG in stoichiometric amounts. However, the lipid A structure of the closely related strain Mesorhizobium huakuii IFO15243 T has been determined ( Choma and Sowinski 2004) it is a unique structure that contains a nonreducing trisaccharide carbohydrate backbone. The model organism Mesorhizobium loti MAFF303099 forms nitrogen-fixing endosymbiosis with the host legume plant Lotus japonicum ( Kaneko et al. To establish the function of LPS in the context of endosymbiosis and pathogenesis, it is advantageous to elucidate both the structure of the LPS and the genes required for its biosynthesis. There are many examples that demonstrate the importance of LPS for the formation of complete nitrogen-fixing symbiosis between bacterial symbionts and their legume host plants ( Carlson et al. The membrane stability and viability of Gram-negative bacteria, as well as the animal innate immune response to LPS, are dependent on the lipid A structure ( Galanos et al. Lipid A, the lipid portion of LPS, anchors the LPS molecule to the outer leaflet of the outer membrane lipid bilayer and is the main focus of this study. Lipopolysaccharide (LPS) is a major cell surface glycoconjugate found in the outer membranes of Gram-negative bacteria and contains a carbohydrate and lipid portion. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis combined with NMR studies revealed that the lipid As from these rgtF-complemented strains were modified with an additional α-(1,1)-GalA attached to the proximal glucosamine.īacterial membrane biosynthesis, lipid A biosynthesis, novel glycosyl transferase Introduction loti rgtF gene into an expression plasmid and introduced that plasmid into Rhizobium etli strains that do not contain the rgtF gene nor produce lipid A α-(1,1)-GalA. In order to determine the predicted rgtF gene function, we cloned the M. We predicted candidate rgtF genes in bacterial species known to produce lipid A with α-(1,1)-GalA. An additional Rhizobium GalAT, RgtE, is required for the biosynthesis of Dod-P-GalA.
The Rhizobium GalAT, RgtD, is the lipid A α-(1–4′)-GalAT which utilizes the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA) for GalA transfer. Species of the Rhizobium genera produce lipid A with α-(1,4′)-GalA but not α-(1,1)-GalA. However, the α-(1,1)-GalA transferase (GalAT) gene, which we have named RgtF, was not identified. loti, the stalk-forming bacterium Caulobacter crescentus and the hyperthermophilic bacterium Aquifex aeolicus. An unusual α-(1,1)-galacturonic acid (GalA) lipid A modification has been reported in the lipopolysaccharide of a number of interesting Gram-negative bacteria, including the nitrogen-fixing bacteria Azospirillum lipoferum, Mesorhizobium huakuii and M.