Cultures were diluted to an OD600 (optical density at 600 nm) of 0.05 in 5 ml of fresh medium and labeled with 32Pi (2.5 μCi/ml; Amersham Biosciences) in N-minimal medium, as indicated. For the time course analysis, cells were first grown in 5 ml of N-minimal containing 10 mM Mg2+ to log phase, washed twice with N-minimal medium, and resuspended with 5 ml of N-minimal medium containing 10 μM Mg2+ and 32Pi (2.5 μCi/ml) for the indicated time. Cells were harvested by centrifugation, and the isolation of 32P-labeled lipid A was carried out by mild acid hydrolysis, as previously described (8, 52). 32P-lipid A species (~1000 cpm·per lane) were analyzed by TLC in a solvent system of chloroform, pyridine, 88% formic acid, and water (50:50:16:5, v/v) and visualized using a PhosphorImager (GE Healthcare).

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