Cell Wall Polysaccharide Biosynthesis by Membrane Fragments from Streptococcus pyogenes and Stabilized L-Form

The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-¹⁴C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of ¹⁴C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.     

Production,isolation and preliminary characterization of the exopolysaccharide of the cyanobacterium Spirulina platensis

Summary The soluble exocellular polysaccharide secreted by the filamentous cyanobacteria Spirulina platensis is a primary metabolite. It is formed by ten different types of monomer units including six neutral sugars (xylose, rhamnose, fucose, galactose, mannose and glucose in the proportions 1.3/0.3/0.7/2.7/traces/2), two unidentified sugars, two uronic acids and sulphate groups accounting for 40 % and 5 % respectively of the mass of the molecule. This polysaccharide displays a non Newtonian behaviour and a strong pseudoplastic characteristic that may originate in the polyelectrolytic property of the molecule.     

Characterization of 2-amino-2,6-dideoxy-D-glucose as a constituent of the lipopolysaccharide antigen of Pseudomonas aeruginosa immunotype 4.

The title lipopolysaccharide was freed from its lipid A component by mild, acid hydrolysis, to give a polysaccharide fraction that was subsequently hydrolyzed completely to afford a mixture of neutral sugars and amino sugars. The amino sugars were separated, and identified as 2-amino-2-deoxy-D-galactose, 2-amino-2,6-dideoxy-galactose as a 2:1 mixture of the D and L enantiomers, and 2-amino-2,6-dideoxy-D-glucose. A reference sample of 2-amino-2,6-dideoxy-D-glucose was synthesized by an improved preparative route. Among the lipopolysaccharide antigens of the seven recognized immunotypes of Pseudomonas aeruginosa, 2-amino-2,6-dideoxyglucose is also characterized as a constituent of two others, types 3 and 5.     

Structure of the O-specific polysaccharide from Shewanella japonica type strain KMM 3299T containing the rare amino sugar Fuc4NAc

An acidic O-specific polysaccharide (PS) of the agar-digesting bacterium Shewanella japonica with the type strain KMM 3299(T) was obtained by mild acid hydrolysis of the lipopolysaccharide. The polysaccharide was studied by component analysis, methylation analysis, (1)H and (13)C NMR spectroscopy, including 2D NMR experiments. The PS was determined to have the following structure involving three unusual amino sugars:     

COCCOLITH-ASSOCIATED POLYSACCHARIDES FROM CELLS OF EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler, a unicellular calcifying alga, consist of calcite closely associated with an acidic, Ca²⁺-binding polysaccharide. This polysaccharide is thought to play a regulatory role in coccolith synthesis by interfering with CaCO₃ crystallization. Here we show that the polysaccharides from three different strains, A 92, L and 92 D, all inhibit the precipitation of CaCO₃ in vitro to the same extent. The monosaccharide compositions of the A 92 and L polysaccharide are similar. The 92 D material, however, deviates from the other two: it contains significantly lower amounts of methylated sugars and ribose, and elevated levels of rhamnose and galactose. It also contains antigenic determinants not detected in the A 92 and L polysaccharides. In contrast to the latter two macromolecules the 92 D polysaccharide migrates as two bands upon polyacrylamide gel electrophoresis, possibly resulting from complexing with small amounts of protein. The coccolith polysaccharide from L cells, cultured at an elevated growth rate, also migrates as two bands. This phenomenon is due to an increase in molecular size distribution. The results suggest that certain properties of the molecule may be subject to variation without interfering with its function.     

COCCOLITH-ASSOCIATED POLYSACCHARIDES FROM CELLS OF EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler, a unicellular calcifying alga, consist of calcite closely associated with an acidic, Ca²⁺-binding polysaccharide. This polysaccharide is thought to play a regulatory role in coccolith synthesis by interfering with CaCO₃ crystallization. Here we show that the polysaccharides from three different strains, A 92, L and 92 D, all inhibit the precipitation of CaCO₃ in vitro to the same extent. The monosaccharide compositions of the A 92 and L polysaccharide are similar. The 92 D material, however, deviates from the other two: it contains significantly lower amounts of methylated sugars and ribose, and elevated levels of rhamnose and galactose. It also contains antigenic determinants not detected in the A 92 and L polysaccharides. In contrast to the latter two macromolecules the 92 D polysaccharide migrates as two bands upon polyacrylamide gel electrophoresis, possibly resulting from complexing with small amounts of protein. The coccolith polysaccharide from L cells, cultured at an elevated growth rate, also migrates as two bands. This phenomenon is due to an increase in molecular size distribution. The results suggest that certain properties of the molecule may be subject to variation without interfering with its function.     

Venom exonuclease. II. Amino acid composition and carbohydrate, metal ion and lipid content in the Crotalus adamanteus venom exonuclease

Exonuclease from Crotalus adamanteus venom has only threonine as N-terminimal amono acid residue. It was examined for its amino acid composition, -SH and S-S groups. It has no free -SH groups and seven S-S bonds. The analysis of the carbohydrate residues in the enzyme proves that it is a glycoprotein. It contains neutral sugars (9.2%), amino sugars (1.9%) and ten sialic acid residues per molecule. The venom exonuclease is a metalloenzyme. This is proven by the existence of Mg2+, Zn2+ and Ca2+ and their specific role in the catalytic reactions. The enzyme contains also triacylglycerols (1.54%) and cholesterol esters (1.43%). The influence of the non-protein moieties of the exonuclease on its catalytic ability has been discussed.     

Purification and characterization of an extracellular polysaccharide from haloalkalophilic Bacillus sp. I-450

During the course of investigation of haloalkalophilic bacteria, we screened some heavily polluted soil samples from the mudflats surrounding the city of Inchon, Korea, for their bioflocculant producing ability. Based on the screening, one isolate no. 450 tentatively identified as Bacillus sp. produced an extracellular polysaccharide having flocculation activity. The isolate produced the polysaccharide during the late logarithmic growth phase. The polymer could be recovered from the supernatant of the fermented medium by cold ethanol precipitation and purified by treating with cetylpyridinium chloride (CPC). The polymer was identified as an acidic polysaccharide containing neutral sugars, namely, galactose, fructose, glucose and raffinose, and uronic acids as major and minor components, respectively. The amount of neutral sugars, uronic acid and amino sugars were 52.4, 17.2 and 2.4%, respectively. The molecular weight of the polysaccharide was found to be 2.2×10⁶ Da. The Fourier transform infrared spectrophotometer (FT-IR) revealed typical characteristics of polysaccharides. ¹H NMR spectrum showed that the polymer is a heteroglycan. Thermogravimetric (TGA) analysis indicated the degradation temperature (T_d) at 290 °C. The rheological analysis of the polymer 450 revealed the pseudoplastic property with shear-thinning effect, while the compression test indicated that the polymer had high gel strength, and the S.E.M. studies showed that the polymer has a porous structure with small pore-size distribution indicating the compactness of the polymer.     

Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide.

An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells. A. tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells. The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria. Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide. Anion-exchange chromatography followed by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar. Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide. Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant. This indicates that the acidic polysaccharide may play a role in the attachment of A. tumefaciens to host soma plant cells.     

O acetylation of the enterobacterial common antigen polysaccharide is catalyzed by the product of the yiaH gene of Escherichia coli K-12

The carbohydrate component of the enterobacterial common antigen (ECA) of Escherichia coli K-12 occurs primarily as a water-soluble cyclic polysaccharide located in the periplasm (ECA(CYC)) and as a phosphoglyceride-linked linear polysaccharide located on the cell surface (ECA(PG)). The polysaccharides of both forms are comprised of the amino sugars N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc). These amino sugars are linked to one another to form trisaccharide repeat units with the structure -->3-alpha-D-Fuc4NAc-(1-->4)-beta-D-ManNAcA-(1-->4)-alpha-D-GlcNAc-(1-->. The hydroxyl group in the 6 position of the GlcNAc residues of both ECA(CYC) and ECA(PG) are nonstoichiometrically esterified with acetyl groups. Random transposon insertion mutagenesis of E. coli K-12 resulted in the generation of a mutant defective in the incorporation of O-acetyl groups into both ECA(CYC) and ECA(PG). This defect was found to be due to an insertion of the transposon into the yiaH locus, a putative gene of unknown function located at 80.26 min on the E. coli chromosomal map. Bioinformatic analyses of the predicted yiaH gene product indicate that it is an integral inner membrane protein that is a member of an acyltransferase family of enzymes found in a wide variety of organisms. The results of biochemical and genetic experiments presented here strongly support the conclusion that yiaH encodes the O-acetyltransferase responsible for the incorporation of O-acetyl groups into both ECA(CYC) and ECA(PG). Accordingly, we propose that this gene be designated wecH.     

Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F

Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocida Type A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1-704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of approximately 10(3) sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [beta(1,4)GlcUA-beta(1,3)GalNAc](n). In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.     

Structure of a Micrococcus lysodeikticus cell wall fragment containing phosphorylated sugars.

A polysaccharide-peptidoglycan complex containing different phosphorylated sugars from Micrococcus lysodeikticus cell wall has been isolated and purified. The peptidoglycan contained muramic acid 6-phosphate and N-acetylglucosamine 6-phosphate as phosphorylated sugars in addition to other sugar residues. Mild acid hydrolysis of the peptidoglycan and subsequent reduction of the released polysaccharide showed therein the presence of glucose and N-acetyl-glucosamine in the linkage of the external polysaccharide residues to the peptidoglycan through phosphodiester linkage. These data suggest the presence of polysaccharide chains linked to a peptidoglycan core through two phosphorylated sugars via two different terminal carbohydrate residues of the external polysaccharide chains in a same polymer.     

Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.     

The action of neutral hypochlorite on epithelial mucopolysaccharides

1. The uptake of dilute neutral hypochlorite by epithelial mucopolysaccharides has been compared with that of proteins, polysaccharides, amino acids and sugars. The uptake has been shown to be related to the protein content of the mucopolysaccharides rather than their polysaccharide content. 2. The destruction of the components of epithelial mucopolysaccharides, certain sugars and polysaccharides after oxidation with dilute neutral hypochlorite at 0-4 degrees has been studied. Very little destruction of the sugar components occurred and in epithelial mucopolysaccharides the only amino acid destroyed specifically was arginine. 3. Oxidation of bovine submaxillary-gland mucoprotein and ovalbumin caused very little destruction of hexosamine and no detectable liberation of this residue as a free reducing group, indicating that the O-seryl galactosaminide and the N-acyl-glycosylamine linkages reported to be present in these compounds were relatively stable to hypochlorite. 4. Depolymerization of epithelial mucopolysaccharides by neutral hypochlorite has been studied by using gel-filtration columns and compared with the depolymerization of polysaccharides and proteins under similar conditions. The polysaccharides examined were relatively resistant to oxidation whereas the proteins were extensively broken down. It is inferred that the extensive depolymerization of epithelial mucopolysaccharides by hypochlorite is related to their protein content rather than their polysaccharide content. 5. Fractionation of the products of oxidation of epithelial mucopolysaccharides by column procedures has revealed that the relative proportions of components in all fractions were similar to those in the original material. 6. Though this study does not provide unequivocal evidence from which the overall structure of this type of epithelial mucopolysaccharide may be deduced, the balance of probabilities now appears to favour a long polypeptide chain to which a large number of oligosaccharide side chains are attached via O-seryl and O-threonyl glycosidic linkages. The results, however, are also consistent with an alternating sequence of short polysaccharide and polypeptide chains and further evidence is necessary before this structure can be ruled out.     

Evidence suggesting that L-fucose is part of a recognition signal for sperm-zona pellucida attachment in mammals

Experiments were designed to test the effects of simple sugars and complex polysaccharides on the attachment of mammalian spermatozoa with the zona pellucida. In the guinea pig, L-fucose was a twofold better inhibitor of the attachment compared to other sugars at 50 mM. Fucoidin, an algal polysaccharide rich in sulfated L-fucose, was a very potent inhibitor, completely blocking attachment at a concentration of 100 μg/ml. Several other highly sulfated glycosaminoglycans showed no inhibitory activity, suggesting the fucoidin effect was not simply due to its charge or sulfate. In addition, fragments of fucoidin, generated by partial hydrolysis and isolated using Biogel P-2, were nearly as inhibitory as the native molecule on a weight basis. Fucoidin also inhibited sperm-zona attachment in the hamster and human; thus, its effect is not species specific. The data suggest that L-fucose may be part of a recognition signal between mammalian gametes.     

Accessibility of lysyl residues of Escherichia coli B/r porin (OmpF) to covalent labeling reagents of different sizes. An approach for a three-dimensional structure of a channel-forming protein

The three-dimensional structure of Escherichia coli B/r porin (OmpF) was studied by chemical modification using activated sugars of different size. Galactose and galactosides of different penetration properties through the porin channel were oxidized by galactose oxidase, and the 6-aldehydes formed were linked to amino groups in porin by reduction with NaBH3CN. Tryptic fragments of modified and unmodified porin were separated by reversed-phase high pressure liquid chromatography and identified by amino acid and amino-terminal analysis from the known primary structure of OmpF. Modification of purified native porin trimers in beta-octylglucoside revealed three classes of amino groups: (i) those not modified by any sugars; (ii) those modified only by small sugars that diffuse rapidly through the pore, such as galactose or melibiose; and (iii) those modified by either small or large sugars, the latter including pore-impermeant sugars such as stachyose. The results suggest that the three classes of amino groups correspond, respectively, to groups buried in the trimeric molecule, those in the interior of the pore and those exposed on the surface of porin. In addition modification experiments performed on whole cells suggested that all the reactive groups modified by the pore-impermeant sugars (class iii) are located on the surface of porin exposed on the outside of the outer membrane.     

The application of 1H/13C inversely correlated NMR spectroscopy to the determination of acylation and glycosylation sites in the O-specific polysaccharide from Hafnia alvei 1187

An inversely correlated 1H/13C NMR spectrum defined the amino sugars acylated by acetyl or 3-hydroxybutyryl groups and revealed partial sequences and glycosylation sites in a tetrasaccharide repeating unit of the title polysaccharide, (----2DGlc alpha 1----3DGlcNAcyl alpha 1----4DGalNAc alpha 1----3DGalNAc beta 1----)n, where Acyl = 3-hydroxybutyryl.     

Assessment of biochemical methods to detect enzymatic depolymerization of polysaccharides

Biochemical methods for detection of depolymerisation were compared. Currently used methods such as reducing sugars assays, double bond monitoring or molecular weight determination were tested to follow the kinetic of depolymerization with different enzyme/polysaccharide combinations. The range of concentrations of different enzymes allowed us to identify the most sensitive and appropriate method to detect polysaccharide degradation. Reducing sugars assays are quantitative, sensitive and usable with all kind of polysaccharide but some compounds may interfere with them. When the polysaccharide is charged, agarose gel electrophoresis, although being a qualitative assay is as sensitive as high performance size exclusion chromatography analysis, easy to handle, high-throughput and this is preferred.     

Golgi Apparatus Mediated Polysaccharide Secretion by Outer Root Cap Cells of Zea mays. III. Control by Exogenous Sugars

Sugars supplied to germinating seedlings of maize (Zea mays L.) regulate the secretion of polysaccharides by the outer cells of the root cap. The polysaccharide secreted by these cells adheres to the root tip as a droplet and the size of the droplet was used to quantitate polysaccharide secretion. The polysaccharide contains glucose, galacrose, and galacturonic acid residues with smaller quantities of mannose, arabinose, xylose, fucose and rhamnose. These sugars supplied to maize seedlings had marked effects on the rate of polysaccharide secretion by root tips. The effects on secretion were independent of the growth rates of the roots. Glucose, fucose and xylose increased droplet size 1.5–2 fold (as did sucrose, maltose, lacrose, fructose and ribose) whereas galactose, arabinose and galacturonic acid were inhibitory. Mannose increased dropler size 5–7 fold. The marked effect of mannose on polysaccharide secretion was due to an increased rate of secretion combined with a longer phase of extrusion of polysaccharide into the forming droplet. The effect of mannose was partially reversed by inorganic phosphate and other sugars (except for fucose which had no effect or promoted secretion in the presence of mannose). In contrast to sucrose, mannose stimulated secretion in a maize variety having a high sugar endosperm (high endogenous sugar). The results suggest that regulation of secretion by mannose is due to an alteration of normal sugar metabolism; whereas stimulation of secretion by sucrose and other sugars may be due to an increased availability of sugars for metabolism.     

Structural studies of capsular polysaccharide of type XII Diplococcus pneumoniae

The capsular polysaccharide of Diplococcus pneumoniae Type XII contains residues of d-glucose and d-galactose in a molar ratio of 2:1. The methylated polysaccharide yielded upon hydrolysis 2,3,4,6-tetra- and 3,4,6-tri-O-methyl-d-glucose and 2,3,4,6-tetra-O-methyl-d-galactose as the only neutral methyl sugars. Periodate oxidation of the polysaccharide resulted in destruction of all neutral sugars and immunochemical activity against rabbit antisera. Periodate oxidation of the methyl O-methylglycosides obtained after hydrolysis of the methylated polysaccharide indicated that at least 30% of the l-fucosamine residues are substituted at C-4 in the polysaccharide. It is concluded that the polysaccharide consists of a hexosamine backbone that is substituted by d-galactosyl and kojibiosyl side-chains. The proposed terminal d-galactosyl residues apparently are sterically hindered from interacting with several d-galactose-binding proteins.