A Phosphoethanolamine Transferase Specific for the Outer 3-Deoxy-D-manno-octulosonic Acid Residue of Escherichia coli Lipopolysaccharide

Abstract

Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo 2 -lipid A. EptB is an inner membrane protein of 574 amino acid residues with five predicted trans-membrane segments within its N-terminal region. An in-frame replacement of eptB with a kanamycin resistance cassette rendered E. coli WBB06 (but not wild-type W3110) hypersensitive to CaCl 2 at 5 mM or higher. Ca 2؉ hypersensitivity was suppressed by excess Mg 2؉ in the medium or by restoring the LPS core of WBB06. The latter was achieved by reintroducing the waaC and waaF genes, which encode LPS heptosyl transferases I and II, respectively. Our data demonstrate that pEtN modification of the outer Kdo protected cells containing heptose-deficient LPS from damage by high concentrations of Ca 2؉ . Based on its sequence similarity to EptA(PmrC), we propose that the active site of EptB faces the periplasmic surface of the inner membrane.The envelope of Gram-negative bacteria consists of an inner membrane (1, 2), a peptidoglycan cell wall (3), and an outer membrane (4). The latter is an asymmetric bilayer with glycerophospholipids on its inner surface and lipopolysaccharide (LPS) 1 on the outside surface (5, 6). LPS consists of three covalently linked portions (6, 7) as follows: 1) the lipid A moiety, a glucosamine-based saccharolipid 2 that serves as the hydrophobic membrane anchor of LPS; 2) the core region, a nonrepeating oligosaccharide modified with phosphate-containing substituents; and 3) the O-antigen, a distal repeating oligosaccharide, which is absent in most strains of Escherichia coli K12 (6). The lipid A moiety and the 3-deoxy-D-manno-octulosonic acid (Kdo) residues of the core are essential for growth of E. coli and most other Gram-negative bacteria (6). Strains lacking all LPS sugars distal to Kdo are termed heptose-deficient or "deep rough" (6, 8). These mutants are viable under laboratory conditions (9), but are hypersensitive to serum and antibiotics (4), and often show reduced virulence in animal models (7).Under certain conditions, strains of E. coli and Salmonella synthesize LPS molecules modified with a phosphoethanolamine (pEtN) group at position 7 of the outer Kdo residue (9) (Fig. 1). Brabetz et al. (9) have reported that E. coli WBB06, which harbors a deletion spanning the heptosyl transferase genes waaC(rfaC) and waaF(rfaF), contains heptose-deficient LPS, modified with pEtN at position 7 of the outer Kdo sugar. Kanipes et al. (10) later demonstrated that pEtN addition to LPS in WBB06 is a consequence of the presence of Ca 2ϩ in the growth medium used by Brabetz et al. (9) and is unrelated to the deletion of the heptosyl transferase genes. A pEtN transferase activity is present in membranes of WBB06 grown in the presence of 5-50 mM Ca 2ϩ (10). The enzyme is stimulated by exogenous phosphatidylethanolamine (PE) and is selective for the outer Kdo residue of Kdo 2 -lipid A and related substrates (10). Addition of EDTA to the in vitro pEtN transferase assay was found to be inhibitory.We have...

Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC

“…6. PmrC is proven to be responsible for the addition of PEtN to the lipid A at the 1-phosphate residue (Lee et al, 2004;Reynolds et al, 2005). Our results have shown that none of the predicted PmrC homologues (Ybip/YhbX/YijP/Ecf3) was involved in the formation of the remaining 1-O-P-PEtN lipid A species.…”

“…Nevertheless, the lipid A profiles clearly indicate expression of PmrC activity, and all other homologues were governed by the same transcriptional and translational start signals. We did not include yhjW (Table 2) because YhjW (EptB) was recently reported to be responsible for the addition of PEtN to the second Kdo residue, but not to lipid A (Reynolds et al, 2005). Interestingly, YijP (CptA) in S. Typhimurium mediates the addition of PEtN to the phosphorylated heptose-I residue (Tamayo et al, 2005).…”

“…Our results have shown that none of the predicted PmrC homologues (Ybip/YhbX/YijP/Ecf3) was involved in the formation of the remaining 1-O-P-PEtN lipid A species. However, YhjW (EptB) was recently reported to be responsible for the addition of PEtN to the second Kdo residue (Reynolds et al, 2005), and YijP (CptA) in S. Typhimurium was involved in the addition of PEtN to the phosphorylated heptose-I residue (Tamayo et al, 2005). E. coli O157 : H7 possesses a plasmid-borne shf locus consisting of shf-wabBecf3-msbB2 (Kim et al, 2004;Kaniuk et al, 2004).…”

“…There are several hypothetical proteins homologous to PmrC in E. coli and S. Typhimurium (Trent & Raetz, 2002;Reynolds et al, 2005). E. coli O157 : H7 possesses a 92 kb plasmid pO157 that carries the shf locus (also known as ecf) consisting of shf-wabB-ecf3-msbB2 (Boerlin et al, 1998;Kim et al, 2004;Yoon et al, 2004;Kaniuk et al, 2004).…”

Glycolipozyme membrane protein integrase (MPIase): recent data

“…However, overproduction of dgkA did not affect the DAG expression, suggesting the occurrence of tight regulation as to DAG expression (17). Several genes are known to be involved in DAG biosynthesis, i.e., mdoB (22,23), pgpB (24)(25)(26), and eptB (27). The products of these genes produce DAG as committed reactions.…”