Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli.
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.
Jia
Wenyi
W
Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.
El Zoeiby
Ahmed
A
Petruzziello
Tania N
TN
Jayabalasingham
Bamini
B
Seyedirashti
Seyedreza
S
Bishop
Russell E
RE
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry; D004187:Disaccharides / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008050:Lipid A / Q000737:chemistry; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D019308:Palmitic Acid / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013696:Temperature; D013997:Time Factors; D001616:beta-Galactosidase / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004187:Disaccharides; D004731:Endotoxins; D008050:Lipid A; D008055:Lipids; D008070:Lipopolysaccharides; C079726:MsbA protein, Bacteria; D009841:Oligonucleotides; D010743:Phospholipids; D019308:Palmitic Acid; D002701:Chloramphenicol; D004247:DNA; D004492:Edetic Acid; D001616:beta-Galactosidase; D008274:Magnesium