FIGURE 2. TLC analysis of ³²P lipid A profiles after mild acid hydrolysis.

Lipid A was labeled with ³²P_i and isolated from cells by mild acid hydrolysis. Wild-type E. coli O157:H7 (4304) and K-12 (MC1061) were analyzed in comparison with their msbB and/or pagP mutant derivatives. Cultures were grown for 150 min and adjusted with or without 25 mM EDTA for an additional 5 min. The isolated lipid A species were separated by TLC and visualized with a PhosphorImager. The main species of lipid A that were identified previously by mass spectrometry are indicated to the left and include the 4′-monophosphate (1-OH), the 1,4′-bis-phosphate (1-OP), the 1-diphosphate (1-O-PP), the 1-diphosphoryl-EtN (1-O-PPEtN), and the 4′-L-Ara4N lipid A species. The penta-, hexa-, and hepta-acylated derivatives of each lipid A species are also indicated to the left (A). Schematic representations (B) reveal the relative migration of certain lipid A molecular subtypes in the TLC plate. The fastest migrating species (hepta-acyl 1-OH) has the greatest number of acyl chains and the least number of polar groups. The acylation patterns for the 1-OH lipid A species are shown as an example, which reveals that the penta-acylated derivative migrates most slowly. Of the two faster migrating hexa-acylated species with 4 + 2 and 3 + 3 acyl chain distributions, the 4 + 2 species migrates perceptibly faster, and this difference becomes accentuated as more polar groups are added. The effect of adding various polar substituents is shown schematically only for the penta-acylated lipid A species, but relative migrations for all combinations of acyl chain distributions and polar substituents can be deduced by extrapolation from these selected examples. Relative migrations are not drawn exactly to scale, and palmitate is shown in red.