Lipopolysaccharide Is a 4-Aminoarabinose Donor to Exogenous Polyisoprenyl Phosphates through the Reverse Reaction of the Enzyme ArnT

Modification of the lipid A portion of LPS with cationic Monosaccharides provides resistance to polymyxins, which are often employed as a last resort to treat multidrug-resistant Bacterial infections. Here, we describe the use of fluorescent polyisoprenoids, liquid chromatography-mass spectrometry, and Bacterial genetics to probe the activity of membrane-localized proteins that utilize the 55-carbon lipid carrier bactoprenyl phosphate (BP). We have discovered that a substantial background reaction occurs when B-strain E. coli cell membrane fractions are supplemented with exogenous BP. This reaction involves proteins associated with the arn operon, which is necessary for the covalent modification of lipid A with the cationic 4-aminoarabinose (Ara4N). Using a series of arn operon gene deletion mutants, we identified that the modification was dependent on ArnC, which is responsible for forming BP-linked Ara4N, or ArnT, which transfers Ara4N to lipid A. Surprisingly, we found that the majority of the Ara4N-modified isoprenoid was due to the reverse reaction catalyzed by ArnT and demonstrate this using heat-inactivated membrane fractions, isolated lipopolysaccharide fractions, and analyses of a purified ArnT. This work provides methods that will facilitate thorough and rapid investigation of Bacterial outer membrane remodeling and the evaluation of polyisoprenoid precursors required for covalent glycan modifications.