Abstract
The biogenesis of biological membranes hinges on the coordinated trafficking of membrane lipids between distinct cellular compartments. The bacterial outer membrane enzyme PagP confers resistance to host immune defenses by transferring a palmitate chain from a phospholipid to the lipid A (endotoxin) component of lipopolysaccharide. PagP is an eight-stranded antiparallel beta-barrel, preceded by an N-terminal amphipathic alpha-helix. The active site is localized inside the beta-barrel and is aligned with the lipopolysaccharide-containing outer leaflet, but the phospholipid substrates are normally restricted to the inner leaflet of the asymmetric outer membrane. We examined the possibility that PagP activity in vivo depends on the aberrant migration of phospholipids into the outer leaflet. We find that brief addition to Escherichia coli cultures of millimolar EDTA, which is reported to replace a fraction of lipopolysaccharide with phospholipids, rapidly induces palmitoylation of lipid A. Although expression of the E. coli pagP gene is induced during Mg2+ limitation by the phoPQ two-component signal transduction pathway, EDTA-induced lipid A palmitoylation occurs more rapidly than pagP induction and is independent of de novo protein synthesis. EDTA-induced lipid A palmitoylation requires functional MsbA, an essential ATP-binding cassette transporter needed for lipid transport to the outer membrane. A potential role for the PagP alpha-helix in phospholipid translocation to the outer leaflet was excluded by showing that alpha-helix deletions are active in vivo. Neither EDTA nor Mg(2+)-EDTA stimulate PagP activity in vitro. These findings suggest that PagP remains dormant in outer membranes until Mg2+ limitation promotes the migration of phospholipids into the outer leaflet.
Highlights
Biological membranes are built from diverse lipids and proteins
We reasoned that EDTA may activate PagP, because it is the only enzyme of lipid A biosynthesis localized in the outer membrane of E. coli [22]
We have demonstrated that brief treatment of growing E. coli with EDTA can induce the palmitoylation of lipid A by the outer membrane enzyme PagP
Summary
Materials—32Pi was purchased from PerkinElmer Life Sciences. Antibiotics, O-nitrophenyl--D-galactoside, and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside were obtained from Sigma. PCR gene amplification was performed with 2.5 units of Pfu polymerase in a volume of 50 l of the supplied buffer with 100 ng of template DNA, 150 ng of the appropriate primers (Table III), and 10 M dNTPs. After initial denaturation for 1 min at 94 °C, 30 cycles of 30 s at 94 °C, 1 min at the appropriate annealing temperature, and 2 min at 72 °C were performed and followed by 10 min at 72 °C. The pellet was washed once with 5 ml of a fresh single-phase Bligh/Dyer mixture, consisting of chloroform/methanol/ water (1:2:0.8, v/v). Partitioning was made by centrifugation, and the lower phase material was collected and washed once with 4 ml of the upper phase derived from a fresh neutral two-phase Bligh/Dyer mixture, consisting of chloroform/methanol/water (2:2:1.8, v/v). The sample was analyzed by TLC as described above to verify that the radioactive material was represented by lipid A
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
