Site-specific acylation changes in the lipid A of Escherichia coli lpxL mutants grown at high temperatures.

LPS is a major component of the outer membrane of Gram-negative bacteria. The lipid A region of LPS mediates stimulation of the immune system. In E. coli, the gene (formerly htrB) codes for a late lauroyltransferase (LpxL) in lipid A biosynthesis. E. coli lpxL mutants have been described previously with impaired growth above 33°C in rich media. However, we were able to grow lpxL mutants at 30°C, 37°C and 42°C, and investigate their lipid A moieties to gain insight into changes and regulatory effects in lipid A biosynthesis. Multiple-stage mass spectrometry was used to decipher unusual lipid A structures produced by lpxL mutant bacteria at high temperatures that rescue the temperature-sensitive phenotype. At 37°C and 42°C, E. coli lpxL mutants appear to activate different acyltransferases or biosynthetic pathways that generate atypical penta- and hexaacyl lipid A structures by incorporating longer fatty acids, such as a secondary palmitoleic acid (2'-O-position, distal) and a secondary palmitic acid (2-O-position, proximal) respectively. However, we observed no changes in these structures through various growth curve stages. This study indicates that E. coli (lpxL) lipid A biosynthesis, and specifically the 'late' acylation of lipid A, is temperature dependent, thus suggesting a highly regulated process.