AimsIdentify the secondary acyltransferases of lipid A in Pseudomonas putida. Methods and ResultsTwo homologues of Escherichia coli acyltransferase LpxL, encoded by PP_0063 and PP_1735, exist in P. putida. Pseudomonas putida mutant strains KWZ001 and KWZ002 were constructed by deleting the genes PP_0063 and PP_1735 respectively. Lipid A species were isolated from P. putidaKT2442, KWZ001 and KWZ002, respectively, and analysed by using electrospray ionization/mass spectrometry (ESI/MS). The results suggest that both PP_0063 and PP_1735 encode secondary acyltransferase of lipid A in P. putida. To further study the site-specificity of these two acyltransferases, PP_0063, PP_1735 and Escherichia coli lpxL were overexpressed in KWZ001 and KWZ002 respectively. Lipid A species isolated from these recombinant strains were analysed by using ESI/MS, and the results suggest that the acyltransferase encoded by PP_0063 catalyses the addition of the 2-OH-C-12:0 chain at the 2-position and the acyltransferase encoded by PP_1735 catalyses the addition of the C-12:0 chain to the 2-position of lipid A in P. putida. ConclusionThe two acyltransferases encoded by PP_0063 and PP_1735, respectively, are responsible for the site-specific additions of the two secondary acyl chains at the 2- and 2-positions of lipid A in P. putida. Significance and Impact of the StudyUnderstanding the lipid A structure variation in P. putida might provide new clues for the survival of P. putida under various stress conditions.