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A hallmark of Francisella tularensis, a highly virulent Gram-negative bacterium, is an unusual LPS that possesses both structural heterogeneity and characteristics that may contribute to innate immune evasion. However, none of the methods yet employed has been sufficient to determine the overall LPS composition of Francisella. We now demonstrate that metabolic labeling of francisellae with [14C]acetate, combined with fractionation of [14C]acetate-labeled lipids by ethanol precipitation rather than hot phenol–water extraction, permits a more sensitive and quantitative appraisal of overall compositional heterogeneity in lipid A and LPS. The majority of lipid A of different francisellae strains grown in diverse bacteriologic media and within human phagocytes accumulated as very hydrophobic species, including free lipid A, with <10% of the lipid A molecules substituted with O-Ag polysaccharides. The spectrum of lipid A and LPS species varied in a medium- and strain-dependent fashion, and growth in THP-1 cells yielded lipid A species that were not present in the same bacteria grown in brain heart infusion broth. In summary, metabolic labeling with [14C]acetate greatly facilitates assessment of the effect of genotypic and/or environmental variables on the synthesis and accumulation of lipid A and LPS by Francisella, including during growth within the cytosol of infected host cells. [ABSTRACT FROM PUBLISHER]