causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of , which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available genomes revealed that this pathogen's genome encodes two orthologues of LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated lipid A, whereas only LpxL2 mediated lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of and expression. LpxL2-dependent lipid A acylation protects from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti- drugs.