Abstract
The synthesis of "typical" hexa-acylated lipid A occurs via a nine-step enzymatic pathway, which is generally well conserved throughout all gram-negative bacteria. One exception to the rule is Helicobacter pylori, which has only eight homologs to the nine lipid A biosynthetic enzymes. The discrepancy occurs toward the end of the pathway, with H. pylori containing only a single putative secondary acyltransferase encoded by jhp0265. In Escherichia coli K-12, two late acyltransferases, termed LpxL and LpxM, are required for the biosynthesis of hexa-acylated lipid A. Detailed biochemical and genetic analyses reveal that H. pylori Jhp0265 (the protein encoded by jhp0265) is in fact an LpxL homolog, capable of transferring a stearoyl group to the hydroxyl group of the 2' linked fatty acyl chain of lipid A. Despite the lack of a homolog to LpxM in the H. pylori genome, the organism synthesizes a hexa-acylated lipid A species, suggesting that an equivalent enzyme exists. Using radiolabeled lipid A substrates and acyl-acyl carrier protein as the fatty acyl donor, we were able to confirm the presence of a second H. pylori late acyl transferase by biochemical assays. After synthesis of the hexa-acylated lipid A species, several modification enzymes then function to produce the major lipid A species of H. pylori that is tetra-acylated. Jhp0634 was identified as an outer membrane deacylase that removes the 3'-linked acyl chains of H. pylori lipid A. Together, this work elucidates the biochemical machinery required for the acylation and deacylation of the lipid A domain of H. pylori lipopolysaccharide.