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. loti, the stalk-forming bacterium Caulobacter crescentus and the hyperthermophilic bacterium Aquifex aeolicus. However, the α-(1,1)-GalA transferase (GalAT) gene, which we have named RgtF, was not identified. Species of the Rhizobium genera produce lipid A with α-(1,4')-GalA but not α-(1,1)-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. An additional Rhizobium GalAT, RgtE, is required for the biosynthesis of Dod-P-GalA. We predicted candidate rgtF genes in bacterial species known to produce lipid A with α-(1,1)-GalA. In order to determine the predicted rgtF gene function, we cloned the M. 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. 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.