Escherichia coli mutants that synthesize dephosphorylated lipid A molecules.

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine that is phosphorylated at the 1 and 4' positions. Expression of the Francisella novicida lipid A 1-phosphatase FnLpxE in E. coli results in dephosphorylation of the lipid A proximal unit. Coexpression of FnLpxE and the Rhizobium leguminosarum lipid A oxidase RlLpxQ in E. coli converts much of the proximal glucosamine to 2-amino-2-deoxygluconate. Expression of the F. novicida lipid A 4'-phosphatase FnLpxF in wild-type E. coli has no effect because FnLpxF cannot dephosphorylate hexaacylated lipid A. However, expression of FnLpxF in E. coli lpxM mutants, which synthesize pentaacylated lipid A lacking the secondary 3'-myristate chain, causes extensive 4'-dephosphorylation. Coexpression of FnLpxE and FnLpxF in lpxM mutants results in massive accumulation of lipid A species lacking both phosphate groups, and introduction of RlLpxQ generates phosphate-free lipid A variants containing 2-amino-2-deoxygluconate. The proposed lipid A structures were confirmed by electrospray ionization mass spectrometry. Strains with 4'-dephosphorylated lipid A display increased polymyxin resistance. Heptose-deficient mutants of E. coli lacking both the 1- and 4'-phosphate moieties are viable on plates but sensitive to CaCl(2). Our methods for reengineering lipid A structure may be useful for generating novel vaccines and adjuvants.