Chemical Characterization of the Lipopolysaccharide of Pseudomonas solanacearum

The carbohydrates present in lipopolysaccharide (LPS) from Pseudomonas solanacearum are rhamnose, xylose, 2-amino-2-deoxyglucose, glucose, heptose, and 2-keto-3-deoxyoctonate. LPS extracted from cultures grown on either glycerol or glucose (as the major source of carbon) and extracted after various incubation periods had similar compositions. The LPS from several strains of the bacterium contained the same component sugars, but the amounts of each sugar varied considerably. It was observed, however, that xylose and 2-amino-2-deoxyglucose increased proportionately with rhamnose, the major component. Phenol-water-extracted LPS contained measurable amounts of nucleic acid, protein, and arabinan, but none of these polymers were detected in LPS extracted with phenol-chloroform-petroleum ether. Polysaccharides liberated from LPS by mild acid hydrolysis were purified by gel filtration. Carbohydrate analysis of the LPS from a virulent, fluidal strain (K60) showed that the O-specific antigen consisted of rhamnose, xylose, and 2-amino-2-deoxyglucose in the proportions 4:1:1. The LPS of an avirulent, afluidal strain (B1) lacked the O-specific antigen; the R-core region consisted of rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate. Methylation analysis indicated that the K60 O-specific antigen was composed of a hexasaccharide repeating unit containing 3-, 2-, and 3,4-substituted rhamnopyranosyl residues, 3-substituted 2-amino-2-deoxyglucose, and terminal xylopyranose in the molar ratios 2:1:1:1:1.     

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.     

Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide

We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an alpha-d-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position.     

Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae.

A sialylated lacto-N-neotetraose (Sial-lNnT) structural unit was identified and structurally characterized in the lipopolysaccharide (LPS) from the genome-sequenced strain Rd [corrected] (RM118) of the human pathogen Haemophilus influenzae grown in the presence of sialic acid. A combination of molecular genetics, MS and NMR spectroscopy techniques showed that this structural unit extended from the proximal heptose residue of the inner core region of the LPS molecule. The structure of the Sial-lNnT unit was identical to that found in meningococcal LPS, but glycoforms containing truncations of the Sial-lNnT unit, comprising fewer residues than the complete oligosaccharide component, were not detected. The finding of sialylated glycoforms that were either fully extended or absent suggests a novel biosynthetic feature for adding the terminal tetrasaccharide unit of the Sial-lNnT to the glycose acceptor at the proximal inner core heptose.     

Identification of a novel inner-core oligosaccharide structure in Neisseria meningitidis lipopolysaccharide.

The structure of the lipopolysaccharide (LPS) from three Neisseria meningitidis strains was elucidated. These strains were nonreactive with mAbs that recognize common inner-core epitopes from meningococcal LPS. It is well established that the inner core of meningococcal LPS consists of a diheptosyl-N-acetylglucosamine unit, in which the distal heptose unit (Hep II) can carry PEtn at the 3 or 6 position or not at all, and the proximal heptose residue (Hep I) is substituted at the 4 position by a glucose residue. Additional substitution at the 3 position of Hep II with a glucose residue is also a common structural feature in some strains. The structures of the O-deacylated LPSs and core oligosaccharides of the three chosen strains were deduced by a combination of monosaccharide analysis, NMR spectroscopy and MS. These analyses revealed the presence of a structure not previously identified in meningococcal LPS, in which an additional beta-configured glucose residue was found to substitute Hep I at the 2 position. This provided the structural basis for the nonreactivity of LPS with these mAbs. The determination of this novel structural feature identified a further degree of variability within the inner-core oligosaccharide of meningococcal LPS which may contribute to the interaction of meningococcal strains with their host.     

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

A partition chromatographic procedure utilizing a cationic exchange resin column in the Li⁺ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of R_e and R_dP^? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R_e or R_dP^? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H⁺ form) released more than 80% of the KDO residues within 15 min. The heptose of the R_dP^? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R_e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R_dP^? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.     

Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation

The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-D-Glcp-(1-->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MSn in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS.     

Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum.

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.     

Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37

The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Identification of a cross-reactive epitope widely present in lipopolysaccharide from enterobacteria and recognized by the cross-protective monoclonal antibody WN1 222-5

Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-alpha-d-Glcp-(1-->3)-[l-alpha-d-Hepp-(1-->7)]-l-alpha-d-Hepp 4P-(1-->3)-R2 (where R1 represents additional sugars of the outer core and R2 represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (KD values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (KD values) determined by SPR were in the range of 3.6 x 10-5 to 3.2 x 10-8 m, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest KD values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5-10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding.     

Identification of a novel structural motif in the lipopolysaccharide of the galE/galK double mutant of Haemophilus influenzae strain Eagan

Defined mutants of the galactose biosynthetic (Leloir) pathway were employed to investigate lipopolysaccharide (LPS) oligosaccharide expression in Haemophilus influenzae type b strain Eagan. The structures of the low-molecular-mass LPS glycoforms from strains with mutations in the genes that encode galactose epimerase (galE) and galactose kinase (galK) were determined by NMR spectroscopy on O- and N-deacylated and dephosphorylated LPS-backbone, and O-deacylated oligosaccharide samples in conjunction with electrospray mass spectrometric, glycose and methylation analyses. The structural profile of LPS glycoforms from the galK mutant was found to be identical to that of the galactose and glucose-containing Hex5 glycoform previously identified in the parent strain [Masoud, H.; Moxon, E. R.; Martin, A.; Krajcarski, D.; Richards, J. C. Biochemistry1997, 36, 2091-2103]. LPS from the H. influenzae strain bearing mutations in both galK and galE (galE/galK double mutant) was devoid of galactose. In the double mutant, Hex3 and Hex4 glycoforms containing di- and tri-glucan side chains from the central heptose of the triheptosyl inner-core unit were identified as the major glycoforms. The triglucoside chain extension, β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, identified in the Hex4 glycoform has not been previously reported as a structural element of H. influenzae LPS. In the parent strain, it is the galactose-containing trisaccharide, β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, and further extended analogues thereof, that substitute the central heptose. When grown in galactose deficient media, the galE single mutant was found to expresses the same population of LPS glycoforms as the double mutant.     

Pseudomonas aeruginosa bacteriophage phi PLS27-lipopolysaccharide interactions

We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations.     

Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme).

The lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype 06 rough-type mutant A28 was isolated by a modified phenol-chloroform-petroleum ether extraction method. Deoxycholate-polyacrylamide gel electrophoresis indicated a single band with mobility similar to that of the complete core region of the wild-type parent serotype 06 (International Antigenic Typing Scheme) strain. Compositional analysis of the LPS indicated that the core oligosaccharide was composed of D-glucose (three units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (two units), 3-deoxy-D-manno-octulosonic acid (two units), L-alanine (one unit), and phosphate (two units). Under the mild conditions of hydrolysis with methanolic hydrogen chloride, a 7-O-carbamoyl substituent was observed on the second heptose residue. The glycan structure of the LPS was determined by employing one- and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry-based methods with a backbone oligosaccharide that was obtained from the LPS by deacylation, dephosphorylation, and reduction of the terminal glucosamine. On the basis of the results of the present study and our earlier work with the P. aeruginosa 06-derived core-defective mutant R5 (H. Masoud, E. Altman, J. C. Richards, and J. S. Lam, Biochemistry, 33:10568-10578, 1994), a structural model for the complete core oligosaccharide is proposed.     

The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein-mediated killing

The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C-reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP-mediated serum bactericidal activity regardless of the genetic background. Differences in CRP-mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.     

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.     

An alternate pattern for globoside oligosaccharide expression in Haemophilus influenzae lipopolysaccharide: structural diversity in nontypeable strain 1124

Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEA-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEA-->4]-alpha-Kdo-(2-->6)-lipid A. We report here structural studies of LPS from nontypeable H. influenzae strain 1124 expressing these motifs linked to both the proximal heptose (HepI) and HepIII at the same time. This novel finding was obtained by structural studies of LPS using NMR techniques and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS(n)() on permethylated dephosphorylated OS. The use of defined mutants allowed us to confirm structures unambiguously and understand better the biosynthesis of each of the globotetraose units. We found that lgtC is involved in the expression of alpha-d-Galp-(1-->4)-beta-d-Galp in both extensions, whereas lic2A directs only the expression of beta-d-Galp-(1-->4)-beta-d-Glcp when linked to HepIII. The LPS of NTHi strain 1124 contained sialylated glycoforms that were identified by CE-ESI-MS/MS. A common sialylated structure in H. influenzae LPS is sialyllactose linked to HepIII. This structure exists in strain 1124. However, results for the lpsA mutant indicate that sialyllactose extends from HepI as well, a molecular environment for sialyllactose in H. influenzae that has not been reported previously. In addition, the LPS was found to carry phosphorylcholine, O-linked glycine, and a third PEA group which was linked to O3 of HepIII.     

Structural analysis of the heptose/hexose region of the lipopolysaccharide from Escherichia coli K-12 strain W3100.

The disaccharide L-glycero-D-manno-heptosyl-D-glucose was isolated from the lipopolysaccharide (LPS) of Escherichia coli K-12 strain W3100 after partial hydrolysis with acid, and the structure was determined by methylation analysis, n.m.r. spectroscopy, and comparison with a synthetic standard. In addition, the oligosaccharides L,D-Hep-D-Glc-D-Glc and L,D-Hep-D-Glc-D-Glc-D-Glc were isolated, and their structures were established by g.l.c.-m.s. and methylation analysis. The results indicated that L-glycero-D-manno-heptose, a characteristic constituent of the inner core region, may also occur in the outer core region which, in E. coli, is generally composed of hexoses. A revised structure of the carbohydrate backbone of the hexose/heptose region of the LPS is given.