Ca2+-induced Phosphoethanolamine Transfer to the Outer 3-Deoxy-d-manno-octulosonic Acid Moiety of Escherichia coli Lipopolysaccharide

Abstract

32 P]lipid IV A , at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mM CaCl 2 , which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mM CaCl 2 suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS. Lipopolysaccharide (LPS)1 is the major constituent of the outer leaflet of the outer membranes of Gram-negative bacteria (1-5). LPS consists of three covalently linked domains. These are: 1) the lipid A moiety, a glucosamine-based phospholipid that serves as the hydrophobic membrane anchor of LPS; 2) the core region, a nonrepeating oligosaccharide decorated with several phosphate-containing substituents; and 3) the O-antigen, a distal repeating oligosaccharide (Fig.

Effect of EDTA on Salmonella enterica serovar Typhimurium involves a component not assignable to lipopolysaccharide release

“…In addition, LPS fractions which are releasable and non-releasable by EDTA should be studied for similar fine structures. In this context it is interesting to note the recent report of Kanipest et al (2001), who demonstrated in E. coli that the critical parameter determining the presence or absence of phosphoethanolamine was the CaCl 2 concentration in the medium. They observed a novel CaCl 2 -induced enzyme that modifies the outer Kdo moiety of E. coli LPS with a phosphoethanolamine group in the presence of 5-50 mM CaCl 2 .…”

An Inner Membrane Dioxygenase that Generates the 2-Hydroxymyristate Moiety of Salmonella Lipid A

“…The addition of a pEtN unit to the outer Kdo residue by EptB (Fig. 1) is independent of PmrA/PmrB and PhoP/ PhoQ, but instead is induced by 5 to 50 mM Ca 2+ ions (22,23).…”

“…The Kdo 2 -lipid A species (unresolved substrate and hydroxylated product) were re-purified from the crude reaction mixture by Bligh/Dyer extraction followed by anion-exchange chromatography on DEAE cellulose (22). The Kdo 2 -lipid A species were then analyzed directly without pH 4.5 hydrolysis by negative ion ESI/MS (Fig.…”

“…The lower phase, which contains the Kdo 2 -lipid A and its hydroxylated product, was removed, washed once with fresh pre-equilibrated acidic upper phase, and then dried under N 2 . The Kdo 2 -lipid A species were then separated from glycerophospholipids by anion exchange chromatography on DEAE cellulose (22), and analyzed by electrospray ionization-mass spectrometry (ESI/MS) in the negative mode.…”

“…The addition of a pEtN unit to the outer Kdo residue by EptB (Fig. 1) is independent of PmrA/PmrB and PhoP/ PhoQ, but instead is induced by 5 to 50 mM Ca 2+ ions (22,23).*Author to whom correspondence should be addressed: C. R. H. Raetz at (919) Fax (919) [33][34][35]. The active sites of these enzymes contain a single ferrous ion, usually coordinated by a His-X-Asp/Glu-X n -His facial triad motif in which n denotes at least 40 consecutive amino acid residues (36).…”