3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a waaA knockout mutation of Escherichia coli

The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae I69 consists of lipid A and a single 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Z?hringer, U. (1988) Eur. J. Biochem. 177, 483-492). The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this strain, and its nucleotide sequence was identical to H. influenzae DSM11121. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and characterized in vitro to encode a monofunctional Kdo transferase. waaA of H. influenzae could not complement a knockout mutation in the corresponding gene of an Re-type Escherichia coli strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-specific Kdo kinase gene (kdkA) of H. influenzae DSM11121 or I69, respectively. The sequences of both kdkA genes were determined and differed in 25 nucleotides, giving rise to six amino acid exchanges between the deduced proteins. Both E. coli strains which expressed waaA and kdkA from H. influenzae synthesized an LPS containing a single Kdo residue that was exclusively phosphorylated at position 4. The structure was determined by nuclear magnetic resonance spectroscopy of deacylated LPS. Therefore, the reaction products of both cloned Kdo kinases represent only one of the two chemical structures synthesized by H. influenzae I69.     

Comparative analyses of secondary gene products of 3-deoxy-D-manno-oct-2-ulosonic acid transferases from Chlamydiaceae in Escherichia coli K-12.

The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci. The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour. The LPS of the mutants were serologically and structurally characterized in comparison to the LPS of the parent strain using compositional analyses, high performance anion exchange chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and specific monoclonal antibodies. The data show that chlamydial Kdo transferases can replace in E. coli K-12 the host's Kdo transferase and retain the product specificities described in their natural background. In addition, we unequivocally proved that WaaA from C. psittaci transfers predominantly four Kdo residues to lipid A, forming a branched tetrasaccharide with the structure alpha-Kdo-(2-->8)-[alpha-Kdo-(2-->4)]-alpha-Kdo-(2-->4)-alpha-Kdo.     

Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins

In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.     

A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum. Purification,substrate specificity,and expression in Salmonella waaC mutants

The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo(2)-lipid IV(A). LpcC containing an N-terminal His(6) tag was assayed using GDP-mannose as the donor and Kdo(2)-[4'-(32)P]lipid IV(A) as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo(2)-[4'-(32)P]lipid IV(A). A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IV(A) or Kdo-lipid IV(A). The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the alpha-anomeric configuration) for the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A). Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner l-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo(2)-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.     

A very efficient method to cleave Lipid A and saccharide components in bacterial lipopolysaccharides.

A novel mild procedure for the selective cleavage of ketosidic linkages is developed using ceric ammonium nitrate (CAN) in anhydrous N,N-dimethylformamide. Its application to lipopolysaccharides (LPS) is very significant because in the so far investigated LPS, the connection between the Lipid A region and the oligo(poly)saccharide part is always a keto-sugar. This procedure has been tested on LPS of Escherichia coli which contains Kdo as a linker between Lipid A and OPS and on Acinetobacter haemoliticus which contains D-glycero-D-talo-2-octulopyranosonic acid (Ko) as a linker and it performed efficiently in both cases.     

Yersinia pestis is viable with endotoxin composed of only lipid A

Lipopolysaccharide (LPS) is the major outer membrane component of gram-negative bacteria. The minimal LPS structure for viability of Escherichia coli and Salmonella enterica serovar Typhimurium is lipid A glycosylated with 3-deoxy-D-manno-octulosonic acid (Kdo) residues. Here we show that another member of the Enterobacteriaceae, Yersinia pestis, can survive without Kdo in its LPS.     

Chlamydial lipopolysaccharide

Chlamydiae are obligatory intracellular parasites which are responsible for various acute and chronic diseases in animals and humans. The outer membrane of the chlamydial cell wall contains a truncated lipopolysaccharide (LPS) antigen, which harbors a group-specific epitope being composed of a trisaccharide of 3-deoxy-D-manno-oct-2-ulosonic (Kdo) residues of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. The chemical structure was established using LPS of recombinant Escherichia coli and Salmonella enterica strains after transformation with a plasmid carrying the gene encoding the multifunctional chlamydial Kdo transferase. Oligosaccharides containing the Kdo region attached to the glucosamine backbone of the lipid A domain have been isolated or prepared by chemical synthesis, converted into neoglycoproteins and their antigenic properties with respect to the definition of cross-reactive and chlamydia-specific epitopes have been determined. The low endotoxic activity of chlamydial LPS is related to the unique structural features of the lipid A, which is highly hydrophobic due to the presence of unusual, long-chain fatty acids.     

A monoclonal antibody against a carbohydrate epitope in lipopolysaccharide differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae, and Chlamydia trachomatis

Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C. pneumoniae, and C. trachomatis in double labeling experiments of infected cell monolayers and by enzyme-linked immunosorbent assay (ELISA). The epitope specificity of mAb S69-4 was determined by binding and inhibition assays using bacteria, LPS, and natural or synthetic Kdo oligosaccharides as free ligands or conjugated to BSA. The mAb bound preferentially Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4)Kdo(2-->4) with a K(d) of 10 microM, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2-->4)Kdo(2-->4) Kdo but not with Kdo(2-->8)Kdo(2-->4)Kdo, Kdo disaccharides in 2-->4- or 2-->8-linkage, or Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin-layer chromatography (TLC) plates in amounts of <10 ng by immunostaining. Due to the high sensitivity achieved in this assay, the antibody also detected in vitro products of cloned Kdo transferases of Chlamydia. The antibody can therefore be used in medical and veterinarian diagnostics, general microbiology, analytical biochemistry, and studies of chlamydial LPS biosynthesis. Further contribution to the general understanding of carbohydrate-binding antibodies was obtained by a comparison of the primary structure of mAb S69-4 to that of mAb S45-18 of which the crystal structure in complex with its ligand has been elucidated recently (Nguyen et al., 2003, Nat. Struct. Biol., 10, 1019-1025).     

A novel 3-deoxy-D-manno-octulosonic acid (Kdo) hydrolase that removes the outer Kdo sugar of Helicobacter pylori lipopolysaccharide

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.     

Molecular basis of lipopolysaccharide heterogeneity in Escherichia coli: envelope stress-responsive regulators control the incorporation of glycoforms with a third 3-deoxy-α-D-manno-oct-2-ulosonic acid and rhamnose

Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes. Examination of LPS heterogeneity revealed overlapping control by RpoE σ factor, two-component systems (BasS/R and PhoB/R), and ppGpp. Deletion of RpoE-specific anti-σ factor rseA led to near-exclusive incorporation of glycoforms with the third Kdo linked to Kdo disaccharide. This was accompanied by concomitant incorporation of rhamnose, linked to either the terminal third Kdo or to the second Kdo, depending upon the presence or absence of phosphoethanolamine on the second Kdo with truncation of the outer core. This truncation in ΔrseA was ascribed to decreased levels of WaaR glycosyltransferase, which was restored to wild-type levels, including overall LPS composition, upon the introduction of rybB sRNA deletion. Thus, ΔwaaR contained LPS primarily with Kdo(3) without any requirement for lipid A modifications. Accumulation of a glycoform with Kdo(3) and 4-amino-4-deoxy-l-arabinose in lipid A in ΔrseA required ppGpp, being abolished in a Δ(ppGpp(0) rseA). Furthermore, Δ(waaZ lpxLMP) synthesizing tetraacylated lipid A exhibited synthetic lethality at 21-23°C pointing to the significance of the incorporation of the third Kdo.     

Mapping of the binding specificity for five monoclonal antibodies recognizing 3-deoxy-D-manno-octulosonic acid in bacterial lipopolysaccharides

Mouse mAb were produced against the deep rough strains Salmonella minnesota R 595, Salmonella typhimurium SL 1102, and Escherichia coli D21f2 and screened by enzyme immunoassay against LPS of several chemotypes. Five antibodies were selected for their ability to bind to chemotype deep rough (Re) LPS which has two 3-deoxy-D-manno-octulosonic acid (Kdo) residues in its nonreducing end. Structurally verified oligosaccharides isolated from rough LPS and synthetic analogues of Kdo were used in an enzyme immunoassay inhibition test to determine the binding epitopes for the antibodies. According to their specificities, the antibodies could be divided into three groups. For two of the groups, the recognized structure was the alpha-Kdo (2----4) Kdo disaccharide and for one group the alpha-Kdo (2----4) alpha-Kdo beta-D-GlcN (1----6) alpha-D-GlcN tetrasaccharide, representing a partial structure of the Re LPS. Inhibition studies with synthetic analogues of Kdo showed that the anomeric configuration and the free carboxyl group of the Kdo residue are important features for antibody binding. Changes in the C-1 to C-6 region of the Kdo molecule do influence the antibody recognition considerably whereas changes in the exocyclic C-7 to C-8 region are of secondary importance. Calculation of the conformation of the inner core region showed that the alpha-Kdo (2----4) alpha-Kdo (2---- disaccharide was free and accessible in chemotype Re LPS, but that linkage of a L-glycero-D-manno-heptose to O-5 of the subterminal Kdo both changes the conformation of the Kdo-disaccharide and covers it thereby making it less accessible.     

A two‐component Kdo hydrolase in the inner membrane of Francisella novicida

Lipid A coats the outer surface of the outer membrane of Gram‐negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2‐keto‐3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residue. F. novicida KdtA is bi‐functional, but F. novicida contains a membrane‐bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co‐transcribed genes. KdoH1 (related to sialidases) has a single predicted N‐terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyses Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non‐ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose‐deficient E. coli mutant that makes Kdo₂‐lipid A as its sole LPS, accumulates Kdo‐lipid A when expressing the both hydrolase components, and 1‐dephospho‐Kdo‐lipid A when expressing both the hydrolase and the Francisella lipid A 1‐phosphatase (LpxE).     

Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-d-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(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.     

The genus-specific lipopolysaccharide epitope of Chlamydia is assembled in C. psittaci and C. trachomatis by glycosyltransferases of low homology

Chlamydiae possess a genus-specific epitope that is located on the lipopolysaccharide (LPS) and is composed of a 3-deoxy-d -manno-octulosonic acid (Kdo) trisaccharide of the sequence αKdo-(2→8)–αKdo–(2→4)-αKdo. In Chlamydia trachomatis, this trisaccharide is biosynthetically generated through the action of a multi-functional Kdo-transferase encoded by the gene gseA. gseA of Chlamydia psittaci 6BC was cloned and expressed in a rough mutant (Re chemotype) of Escherichia coli (strain F515) that contains an LPS with only two α2→4-linked Kdo residues. Recombinant strains were able to add the immunodominant Kdo residue in a α2→8-linkage to the parental LPS, as determined by SDS–PAGE and Western blot analysis using a monoclonal antibody against the genus-specific epitope. The DNA sequence of gseA was determined and aligned to that published recently for C. trachomatis serovar L2. Most surprisingly, the two deduced amino acid sequences shared only an overall homology of 67%. Thus, gseA exhibits species specificity at the DNA level, whereas its gene product results in the synthesis of a carbohydrate antigen with genus specificity.     

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.     

Lipopolysaccharide biosynthesis without the lipids: recognition promiscuity of Escherichia coli heptosyltransferase I

Heptosyltransferase I (HepI) is responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-D-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k(cat)/K(m) of 2.7 × 10(6) M(-1) s(-1), compared to a value of 2.2 × 10(5) M(-1) s(-1) for the Kdo(2)-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates.     

A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant

Addition of a phosphoethanolamine (pEtN) moiety to the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of lipopolysaccharide (LPS) in WBB06, a heptose-deficient Escherichia coli mutant, occurs when cells are grown in 5-50 mM CaCl2 (Kanipes, M. I., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 1156-1163). A Ca2+-induced, membrane-bound enzyme was responsible for the transfer of the pEtN unit to the Kdo domain. We now report the identification of the gene encoding the pEtN transferase. E. coli yhjW was cloned and overexpressed, because it is homologous to a putative pEtN transferase implicated in the modification of the beta-chain heptose residue of Neisseria meningitidis lipo-oligosaccharide (Mackinnon, F. G., Cox, A. D., Plested, J. S., Tang, C. M., Makepeace, K., Coull, P. A., Wright, J. C., Chalmers, R., Hood, D. W., Richards, J. C., and Moxon, E. R. (2002) Mol. Microbiol. 43, 931-943). In vitro assays with Kdo2-4'-[32P]lipid A as the acceptor showed that YhjW (renamed EptB) utilizes phosphatidylethanolamine in the presence of Ca2+ to transfer the pEtN group. Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo2-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 CaCl2 at 5 mM or higher. Ca2+ hypersensitivity was suppressed by excess Mg2+ 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 Ca2+. Based on its sequence similarity to EptA(PmrC), we propose that the active site of EptB faces the periplasmic surface of the inner membrane.     

A novel 3-deoxy-D-manno-octulosonic acid transferase from Chlamydia trachomatis required for expression of the genus-specific epitope.

DNA cloned from Chlamydia trachomatis is able to direct the formation of the genus-specific lipopolysaccharide epitope of chlamydiae in enteric Gram-negative bacteria. We now demonstrate that a single C. trachomatis gene (gseA) is sufficient to impart this trait to Escherichia coli. The deduced amino acid sequence of gseA shows 23% identity (66% similarity) to kdtA, an E. coli gene that codes for a bifunctional enzyme catalyzing the addition of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A precursors (Clementz, T., and Raetz, C. R. H. (1991) J. Biol. Chem. 266, 9687-9696). Extracts of E. coli expressing gseA transfer at least one additional Kdo unit from CMP-Kdo to precursors already bearing the two Kdo residues attached by the kdtA gene product. Introduction of gseA into an E. coli mutant with a thermolabile kdtA gene product endows cell extracts with the ability to transfer not only the third but also the first two Kdos to lipid A precursors, demonstrating that the C. trachomatis enzyme is at least trifunctional. Given the similarities of these two Kdo transferases and the essentiality of Kdo in Gram-negative bacteria, lipopolysaccharide biosynthesis may be a target for development of novel drugs effective against chlamydiae.     

A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic acid (Kdo) kinase. Possible involvement of kdo phosphorylation in bacterial virulence.

The lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue derivatized with either a phosphate or an ethanolamine pyrophosphate moiety at the 4-OH position. In previous studies, we identified a kinase unique to H. influenzae extracts that phosphorylates Kdo-lipid IV(A), a key precursor of lipopolysaccharide in this organism. We have now identified the gene encoding the Kdo kinase by using an expression cloning approach. A cosmid library containing random DNA fragments from H. influenzae strain Rd was constructed in Escherichia coli. Extracts of 472 colonies containing individual hybrid cosmids were assayed for Kdo kinase activity. A single hybrid cosmid directing expression of the kinase was found. The kinase gene was identified by activity assays, sub-cloning, and DNA sequencing. When the putative kinase gene was expressed in E. coli behind a T7 promoter, massive overproduction of kinase activity was achieved ( approximately 8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IV(A) as the substrate, were the same as observed with H. influenzae membranes. Unexpectedly, the kinase gene was identical to a previously characterized open reading frame (orfZ), which had been shown to be important for establishing bacteremia in an infant rat model (Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbiol. 22, 951-965). However, based solely on the genome sequence of H. influenzae Rd, no biochemical function had been assigned to the product of orfZ, which we now designate kdkA ("Kdo kinase A"). Although Kdo phosphorylation may be critical for bacterial virulence of H. influenzae, it does not appear to be required for growth.     

Evidence for a two-metal-ion mechanism in the cytidyltransferase KdsB, an enzyme involved in lipopolysaccharide biosynthesis

Lipopolysaccharide (LPS) is located on the surface of Gram-negative bacteria and is responsible for maintaining outer membrane stability, which is a prerequisite for cell survival. Furthermore, it represents an important barrier against hostile environmental factors such as antimicrobial peptides and the complement cascade during Gram-negative infections. The sugar 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an integral part of LPS and plays a key role in LPS functionality. Prior to its incorporation into the LPS molecule, Kdo has to be activated by the CMP-Kdo synthetase (CKS). Based on the presence of a single Mg²⁺ ion in the active site, detailed models of the reaction mechanism of CKS have been developed previously. Recently, a two-metal-ion hypothesis suggested the involvement of two Mg²⁺ ions in Kdo activation. To further investigate the mechanistic aspects of Kdo activation, we kinetically characterized the CKS from the hyperthermophilic organism Aquifex aeolicus. In addition, we determined the crystal structure of this enzyme at a resolution of 2.10 Å and provide evidence that two Mg²⁺ ions are part of the active site of the enzyme.