Isolation and Characterization of Nonconverting Mutants of Bacteriophage ε34

epsilon(34) Is a converting bacteriophage which brings about a glucosylation of the O antigen in the E group salmonella, thus producing antigen 34. Uridine diphosphate glucose is the precursor of antigen 34 and a glucosyl-lipid is an essential intermediate in this process. Mutants of epsilon(34) which cannot produce antigen 34 have been isolated. Cells lysogenic for these mutants are of two types. Type 1 mutant lysogens cannot form the glucosyl-lipid intermediate; those of type 2 can form glucosyl-lipid but are unable to transfer glucose from the lipid to the O antigen.     

Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O164 cross-reacting with Shigella dysenteriae type 3.

The structure of the O-antigen polysaccharide from Escherichia coli O164 has been determined. Nuclear magnetic resonance spectroscopy together with component and methylation analyses of lipid free polysaccharide were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity NMR experiments. It is concluded that the polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [structure: see text]. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on intact lipopolysaccharide and from the resulting molecular mass, the O-antigen part was estimated to contain approximately 24 repeating units. The nature of the previously reported cross-reactivity of this O-antigen to those of Escherichia coli O124 and Shigella dysenteriae type 3 is discussed.     

The structure of pneumococcal lipoteichoic acid. Improved preparation, chemical and mass spectrometric studies.

Pneumococcal lipoteichoic acid was extracted and purified by a novel, quick and effective procedure. Structural analysis included methylation, periodate oxidation, Smith degradation, oxidation with CrO3, and fast-atom-bombardment mass spectrometry. Hydrolysis with 48% (by mass) HF and subsequent phase partition yielded the lipid anchor (I), the dephosphorylated repeating unit of the chain (II) and a cleavage product of the latter (III). The proposed structures are: (I) Glc(beta 1----3)AATGal(beta 1----3)Glc(alpha 1----3)acyl2Gro, (II) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc(beta 1----1)ribitol and (III) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxygalactose, and all sugars are in the pyranose form and belong to the D-series. Alkaline phosphodiester cleavage of lipoteichoic acid, followed by treatment with phosphomonoesterase, resulted in the formation of II and IV, with IV as the prevailing species: [sequence: see text] The linkage between the repeating units was established as phosphodiester bond between ribitol 5-phosphate and position 6 of the glucosyl residue of adjacent units. The chain was shown to be linked to the lipid anchor by a phosphodiester between its ribitol 5-phosphate terminus and position 6 of the non-reducing glucosyl terminus of I. The lipoteichoic acid is polydisperse: the chain length may vary between 2 and 8 repeating units and variations were also observed for the fatty acid composition of the diacylglycerol moiety. Preliminary results suggest that repeating units II and IV are enriched in separate molecular species. All species were associated with Forssman antigenicity, albeit to a various extent when related to the non-phosphocholine phosphorus. Owing to its unique structure, the described macroamphiphile may be classified as atypical lipoteichoic acid.     

Structural studies on the minor teichoic acid of Bacillus coagulans AHU 1631

The minor teichoic acid linked to glycopeptide was isolated from lysozyme digests of Bacillus coagulans AHU 1631 cell walls, and the structure of the teichoic acid moiety and its junction with the peptidoglycan were studied. Hydrolysis of the teichoic-acid--glycopeptide complex with hydrogen fluoride gave a nonreducing oligosaccharide composed of glucose, galactose and glycerol in a molar ratio of 3:1:1 which was presumed to be dephosphorylated repeating units of the polymer chain. From the results of structural analysis involving NaIO4 oxidation, methylation and acetolysis, the above fragment was characterized as glucosyl(beta 1----3)glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol. In addition, the Smith degradation of the complex yielded a phosphorus-containing fragment identified as glycerol-P-6-glucosyl(beta 1----1/3)glycerol. These results led to the most likely structure for the repeating units of the teichoic acid, -6[glucosyl(beta 1----3)]glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol-P-. The minor teichoic acid, just like the major teichoic acid bound to the linkage unit, was released by heating the cell walls at pH 2.5. The mild alkaline hydrolysis of the minor teichoic acid after reduction with NaB3H4 gave labeled saccharides characterized as glucosyl(beta 1----6)galactitol and glucosyl(beta 1----3)glucosyl(beta 1----6)galactitol, together with a large amount of the unlabeled repeating units of the teichoic acid chain. Thus, the minor teichoic acid chain is believed to be directly linked to peptidoglycan at the galactose residue of the terminal repeating unit without a special linkage sugar unit.     

Long-Chain Glycerol Diether and Polyol Dialkyl Glycerol Triether Lipids of Sulfolobus acidocaldarius

Cells of Sulfolobus acidocaldarius contain about 2.5% total lipid on a dry-weight basis. Total lipid was found to contain 10.5% neutral lipid, 67.6% glycolipid, and 21.7% polar lipid. The lipids contained C(40)H(80) isopranol glycerol diethers. Almost no fatty acids were present. The glycolipids were composed of about equal amounts of the glycerol diether analogue of glucosyl galactosyl diglyceride and a glucosyl polyol glycerol diether. The latter compound contained an unidentified polyol attached by an ether bond to the glycerol diether. The polar lipids contained a small amount of sulfolipid, which appeared to be the monosulfate derivative of glucosyl polyol glycerol diether. About 40% of the lipid phosphorus was found in the diether analogue of phosphatidyl inositol. The remaining lipid phosphorus was accounted for by approximately equal amounts of two inositol monophosphate-containing phosphoglycolipids, inositolphosphoryl glucosyl galactosyl glycerol diether and inositolphosphoryl glucosyl polyol glycerol diether.     

Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases

An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases.     

The polysaccharides from heterocyst and spore envelopes of a blue-green alga. Structure of the basic repeating unit.

The polysaccharides from the envelopes of heterocysts and spores of Anabaena cylindrica consist of repeating units containing 1 mannosyl and 3 glucosyl residues, all linked by beta(1 yields 3) glycosidic bonds, with glycosidic bonds, with glucose, xylose, galactose, and mannose present in side branches. Degradation of the polysaccharides with specific glycosidases has permitted identification of the linkages to almost all of the branches. When the polysaccharides, from which all but two types of side branches had been cleaved, were digested with a beta(1 yields 3) endoglucanase, glucose, a tri-, and a pentasaccharide were produced. The oligosaccharide products were identified as (see article of journal). The backbones of the polysaccharides were sequenced from the reducing terminus by a modified Smith degradation. Analysis with NaB3H4 at each stage of the degradation showed that the backbones terminate in the sequence Man-Glc-Glc-Glc and are therefore presumed to have the structure (Man-Glc-Glc-Glc)n, and that they contain an average of from 128 to 150 sugar residues. From the information obtained, the repeating sequences of the original polysaccharides from the two types of differentiated cells of A. cylindrica could be largely deduced and appeared to be identical.     

A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.Shigella flexneri is a pathovar of Escherichia coli that is the main causative agent of endemic bacillary dysentery (shigellosis). It is estimated that S. flexneri is responsible for approximately 100 million shigellosis cases annually, resulting in hundreds of thousands of deaths, predominantly in young children (11). Currently no vaccine is available, although there is evidence to suggest that serotype-specific immunity occurs following infection and that induction of immunity can be replicated with vaccines (9). Shigella serotype diversity arises due to differences in the chemical structure of the O-antigen repeating unit in the lipopolysaccharide, which is the main target of the adaptive host immune response following infection.Because immunity to S. flexneri can be conferred by the induction of antibodies directed against the O antigen, an understanding of the prevalence of different serotypes and the underlying basis of serotype diversity can inform appropriate vaccine design. All S. flexneri serotypes (with the exception of serotype 6) share a common O-antigen backbone, consisting of a repeating tetrasaccharide unit that is comprised of one N-acetylglucosamine residue (GlcNAc) and three rhamnose residues (RhaI, RhaII, and RhaIII) (14). The 12 traditionally recognized S. flexneri serotypes differ by the presence or absence of just six different chemical modifications (glucosylations or O acetylations) of the O antigen. The genes responsible for these O-antigen modifications are introduced into the bacterial genome via bacteriophages (3). Glucosylation of the S. flexneri O antigen is mediated by three genes [gtrA, gtrB, and gtr(type)] that are arranged in a single operon known as a gtr cluster. gtrA and gtrB are highly conserved between different gtr clusters and encode proteins involved in transferring the glucosyl group from the cytoplasm into the periplasm, where O-antigen modification is thought to take place. gtr(type) is unique to each gtr cluster and encodes a glucosyltransferase that is responsible for attaching the glucosyl group to a specific sugar unit of the O antigen via a specific linkage (3).Investigations of S. flexneri have typically focused on serotypes for which commercially available typing sera are available. More recently, it has become clear that other serotypes are also epidemiologically important. In Bangladesh in the late 1980s, two novel S. flexneri strains that did not agglutinate with antibodies specific for the traditionally recognized serotypes were isolated (4). Chemical analysis of the O antigen revealed that these strains belonged to a new serotype, which was named serotype 1c due to the similarity its O antigen shares with the O antigens of serotype 1a and 1b strains (19). Serotype 1c has since been isolated in Egypt, Indonesia, Pakistan, and Vietnam (6, 15, 18). Serotype 1c was shown to be the most prevalent S. flexneri serotype in a northern province of Vietnam, accounting for more than a third of all S. flexneri strains isolated from 1998 to 1999 (15). Identification of serotype 1c currently relies on agglutination testing using monoclonal antibody MASF Ic (19).The O antigen of serotype 1c is distinguished by the presence of a disaccharide (two glucosyl groups) linked to the GlcNAc in the tetrasaccharide repeating unit of the O antigen. The first glucosyl group is joined to GlcNAc via an α1→4 linkage, as occurs in the O antigen of serotype 1a and serotype 1b strains (type I modification). The O antigen of serotype 1c is distinguished by the presence of a second glucosyl group that is linked to the first via an α1→2 linkage (Fig. ​(Fig.1).1). Type Ia modification is prerequisite to type Ic modification.Open in a separate windowFIG. 1.Chemical structure of the tetrasaccharide repeat units in the O antigens of S. flexneri serotypes 1a and 1c. Note that the O antigen of serotype 1b (not shown) differs from that of serotype 1a by the O acetylation of l-RhaIII.In this study, the genetic basis of O-antigen modification in serotype 1c was elucidated. Serotype 1c strains isolated from different locations and times were compared to gain insight into the evolution of this serotype. This is the first report of the identification of a glucosyltransferase gene that is responsible for addition of the second glucosyl group, causing serotype conversion from serotype 1a to serotype 1c.     

Structure of a capsular polysaccharide isolated from Salmonella enteritidis

Salmonella enteritidis is a food-borne enteric human pathogen that can form a complex protective extracellular matrix. We describe here a component of this matrix which is distinct from other known salmonella extracellular polysaccharides such as cellulose and colanic acid. We have used glycosyl composition and linkage analysis, as well as 1D and 2D NMR spectroscopy to determine the structure of this polysaccharide. We propose that the primary saccharide in the S. enteritidis capsule has a branched tetrasaccharide repeating unit having the following structure: -->3)-alpha-D-Galp-(1-->2)-[alpha-Tyvp-(1-->3)]-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->. This structure is partially substituted on both tyvelose and galactose with a glucose-containing side chain. It further bears considerable similarity to the O antigen from this organism, a feature found in a number of other capsules from Gram-negative bacteria. In addition, we have detected fatty acids at levels that indicate the presence of a lipid anchor.     

The role of glucolipid in the biosynthesis of glycoprotein in Tetrahymena pyriformis

Cell-free preparations from Tetrahymena pyriformis catalyze the incorporation of glucose from UDP-glucose into a glucolipid having properties which are identical to those of other dolichyl phosphoryl sugar derivatives. Kinetic and other experiments have provided evidence that this glucolipid serves as glucose donor for two other types of glucosylated substances, one of which has been tentatively identified as an oligosaccharide lipid and the other a glycoprotein or glycoproteins. In addition, the partially purified glucolipid served as a glucosyl donor to these cell components, suggesting that in this protozoan, at least part of the glycoprotein is synthesized by reactions involving lipid-linked sugars in a manner analogous to that which has been observed in glycoprotein synthesis in mammalian cells.     

The Klebsiella O3 lipopolysaccharide isolated from culture fluid: structure of the polysaccharide moiety

In a series of our earlier studies, the O3 antigen isolated from culture supernatant of Klebsiella pneumoniae strain Kasuya (O3:K1) (KO3) was shown to exhibit very strong adjuvant activity in mice. KO3 obtained was homogeneous in analyses by either gel filtration or ultracentrifugation. Its molecular weight determined by ultracentrifugal analysis was greater than 2 X 10(6). It contained 37.9% C, 6.20% H, 0.24% N, and less than 0.1% P. KO3 was degraded into the polysaccharide moiety and lipid moiety (about 20%) by hydrolysis with 1% acetic acid at 100 C for 1 hr. The molecular weight of the polysaccharide moiety obtained by the hydrolysis was 16,200 as determined by the Somogyi-Nelson method. Chemical analyses using methylation analysis and Smith degradation as the principal methods indicated that the polysaccharide moiety consisted of a mannan which has a pentasaccharide repeating unit of alpha-mannosyl-1,3-alpha-mannosyl-1,2-alpha-mannosyl-1,2-alpha-mannosyl-1, 2-alpha-mannose joined through alpha-1,3-mannosyl linkages. The number of repetitions was less than 20. The fact that minor components such as 2-keto-3-deoxyoctonate and glucose were detected suggests the presence of a core oligosaccharide, but its precise structure is unknown.     

Liposomes containing glucosyl ceramide specifically bind T4 bacteriophage: a self-assembling nanocarrier formulation

A unique formulation is described comprising liposomes containing glucosyl ceramide (GluCer) in the lipid bilayer to which bacteriophage T4 was attached. Binding of the phage T4 did not occur to glycolipids, such as galactosyl ceramide, containing an aldose in which the C-2 or C-4 conformations were not identical to glucose. These results strongly support previous proposals that glucose is a major receptor moiety for T4 binding to Escherichia coli. By using the binding of T4 to liposomal GluCer, we further describe a formulation that can be used as a self-assembling combined antigen and adjuvant carrier. A peptide antigen derived from C-trimer (Ct) of HIV-1 gp41 was fused to the highly antigenic outer capsid protein (Hoc), a nonessential protein of T4 that spontaneously binds to the T4 capsid. This resulted in display of the Ct-Hoc construct on the T4 capsid, and specific binding of a human monoclonal antibody that recognizes a peptide sequence of Ct was demonstrated. Liposomes containing monophosphoryl lipid A (MPLA) have been demonstrated to have potent adjuvant activities for experimental vaccines both in humans and animals, and because of this, mice were immunized with the Ct-Hoc-T4 construct that was bound to liposomes containing both GluCer and MPLA, resulting in the induction of high titers of Ct-specific antibodies. We conclude that liposomes containing both GluCer and MPLA can spontaneously bind to a construct of T4 that displays antigens that spontaneously binds to the capsid of T4 bacteriophage. This formulation could be utilized as an easily manufactured self-assembling antigen and adjuvant carrier.     

Identification of a second lipopolysaccharide in Porphyromonas gingivalis W50

We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. (1)H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.     

Cloning and analysis of the glucosyl transferase gene encoding type I antigen in Shigella flexneri

The O-antigen of most Shigella flexneri serotypes contains an identical tetrasaccharide repeating unit. Apart from serotype Y, the O-antigen is modified by addition of a glucosyl and/or O-acetyl residue to a specific position in the O-unit. In this study the glucosyl transferase gene from a serotype 1a has been cloned and identified. The bacteriophage SfV integrase (int) gene was used to probe a S. flexneri Y53 (serotype 1a) cosmid library and 18 unique clones were identified. Southern hybridisation of these clones indicated two unlinked regions of the chromosome contained the int homologue. When expressed in a live candidate vaccine strain of S. flexneri serotype Y (SFL124), clones with one region produced type I antigen, whereas clones containing the other region produced mainly type Y antigen. One of the cosmid clones positive for type I antigen by agglutination and Western blotting was selected for further study. Genes involved in O-antigen glucosyl modification were mapped on a 5.8 kb fragment and subclones were produced which fully or partially expressed the type I antigen, depending on the extent of the clone. Fully and partially expressing clones may be useful vaccine candidate strains for protection against disease caused by two serotypes of S. flexneri.     

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis.

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.     

A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.     

Glycosidases in organic solvents: I. Alkyl-β-glucoside synthesis in a water-organic two-phase system

A low-water organic solvent two-phase system suitable for glycosylation of hydrophobic substrates is described. Almond β-glucosidase adsorbed on polymeric supports has been shown to catalyse alkyl-β-glucoside synthesis via a transferase reaction or through direct condensation of the glucosidic bond. High concentrations of glucosyl donors were present in the aqueous phase, while water-immiscible primary alcohols, which form the organic phase, served as acceptors of glucose. Reaction yield appeared to be thermodynamically controlled. The influence of various support materials, glucosyl donors, and glucosyl acceptors on reaction rate and product yield was investigated.     

The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase

Rice BGlu1 β-glucosidase is an oligosaccharide exoglucosidase that binds to six β-(1→4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH 5, consistent with the role of E176 as the catalytic acid–base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q, its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90 Å resolution, respectively. The Q176 Nε was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its O2 and Q176 Oε and O1 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration.     

A new model of pneumococcal lipoteichoic acid structure resolves biochemical, biosynthetic, and serologic inconsistencies of the current model

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(beta1-->3)AATGal(beta1-->3)Glc(alpha1-->3)-acyl(2)Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(alpha1-->3)GalNAc(beta1-->, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(alpha1-->3)-acyl(2)Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.     

Structural and genetic characterization of the Shigella boydii type 13 O antigen

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coli constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coli and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected 1H, 13C and 1H, 31P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.