The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens. Heterologous expression of lipid A modification enzymes may enable the development of new vaccines.

The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. In a number of pathogenic Gram-negative bacteria, PagP confers resistance to certain cationic antimicrobial peptides produced during the host innate immune response. The global fold of Escherichia coli PagP was determined in both dodecylphosphocholine and n-octyl-␤-D-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel ␤-barrel preceded by an amphipathic ␣ helix. The ␤-barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual ␤-strands. Three amino acid residues critical for enzymatic activity localize to extracellular loops near the membrane interface, positioning them optimally to interact with the polar headgroups of lipid A. Hence, the active site of PagP is situated on the outer surface of the outer membrane. Because the phospholipids that donate palmitate in the enzymatic reaction are normally found only in the inner leaflet of the outer membrane, PagP activity may depend on the aberrant migration of phospholipids into the outer leaflet. This finding is consistent with an emerging paradigm for outer membrane enzymes in providing an adaptive response toward disturbances in the outer membrane.

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C-terminal His(6)-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of Yersinia and Bordetella species. PagP may provide an adaptive response toward both Mg(2+) limitation and host innate immune defenses.

The ability of enzymes to distinguish between fatty acyl groups can involve molecular measuring devices termed hydrocarbon rulers, but the molecular basis for acyl-chain recognition in any membrane-bound enzyme remains to be defined. PagP is an outer membrane acyltransferase that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from a phospholipid to lipid A (endotoxin). PagP can distinguish lipid acyl chains that differ by a single methylene unit, indicating that the enzyme possesses a remarkably precise hydrocarbon ruler. We present the 1.9 Å crystal structure of PagP, an eight-stranded β-barrel with an unexpected interior hydrophobic pocket that is occupied by a single detergent molecule. The buried detergent is oriented normal to the presumed plane of the membrane, whereas the PagP β-barrel axis is tilted by approximately 25°. Acyl group specificity is modulated by mutation of Gly88 lining the bottom of the hydrophobic pocket, thus confirming the hydrocarbon ruler mechanism for palmitate recognition. A striking structural similarity between PagP and the lipocalins suggests an evolutionary link between these proteins

Significance The outer membrane (OM) of Gram-negative bacteria is composed of lipopolysaccharide (LPS) and glycerophospholipids (GPLs). Environmental regulation of LPS promotes bacterial resistance to host cationic antimicrobial peptides by altering surface charge and hydrophobicity. This work demonstrates that pathogenic Salmonella coordinately regulate GPL and LPS through the PhoPQ regulatory proteins and the OM palmitoyltransferase enzyme PagP. The broad conservation of PhoPQ and PagP in bacteria suggests that environmental regulation of OM GPL may be a conserved stress-response strategy for antimicrobial resistance. Improved understanding of OM GPL synthesis, transport, and modification could lead to new therapies that target the bacterial OM barrier.