Comparative Study of Immunogenic Properties of Purified Capsular Polysaccharides from Streptococcus suis Serotypes 3, 7, 8, and 9: the Serotype 3 Polysaccharide Induces an Opsonizing IgG Response.

Streptococcus suis is an encapsulated bacterium and one of the most important swine pathogens and a zoonotic agent for which no effective vaccine exists. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. In addition to the previously known serotypes 2 and 14, which are nonimmunogenic, we have recently purified and described the CPS structures for serotypes 1, 1/2, 3, 7, 8, and 9. Here, we aimed to elucidate how these new structurally diverse CPSs interact with the immune system to generate anti-CPS antibody responses. CPS-stimulated dendritic cells produced significant levels of C-C motif chemokine ligand 3 (CCL3), partially via Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88-dependent pathways, and CCL2, via TLR-independent mechanisms. Mice immunized with purified serotype 3 CPS adjuvanted with TiterMax Gold produced an opsonizing IgG response, whereas other CPSs or adjuvants were negative. Mice hyperimmunized with heat-killed S. suis serotypes 3 and 9 both produced anti-CPS type 1 IgGs, whereas serotypes 7 and 8 remained negative. Also, mice infected with sublethal doses of S. suis serotype 3 produced primary anti-CPS IgM and IgG responses, of which only IgM were boosted after a secondary infection. In contrast, mice sublethally infected with S. suis serotype 9 produced weak anti-CPS IgM and IgG responses following a secondary infection. This study provides important information on the divergent evolution of CPS serotypes with highly different structural and/or biochemical properties within S. suis and their interaction with the immune system.

Explaining the Serological Characteristics of Streptococcus suis Serotypes 1 and 1/2 from Their Capsular Polysaccharide Structure and Biosynthesis.

The capsular polysaccharide (CPS) is a major virulence factor in many encapsulated pathogens, as it is the case for Streptococcus suis, an important swine pathogen and emerging zoonotic agent. Moreover, the CPS is the antigen at the origin of S. suis classification into serotypes. Hence, analyses of the CPS structure are an essential step to dissect its role in virulence and the serological relations between important serotypes. Here, the CPSs of serotypes 1 and 1/2 were purified and characterized for the first time. Chemical and spectroscopic data gave the following repeating unit sequences: [6)[Neu5Ac(α2-6)GalNAc(ß1-4)GlcNAc(ß1-3)]Gal(ß1-3)Gal(ß1-4)Glc(ß1-]n (serotype 1) and [4)[Neu5Ac(α2-6)GalNAc(ß1-4)GlcNAc(ß1-3)]Gal(ß1-4)[Gal(α1-3)]Rha(ß1-4)Glc(ß1-]n (serotype 1/2). The Sambucus nigra lectin, which recognizes the Neu5Ac(α2-6)Gal/GalNAc sequence, showed binding to both CPSs. Compared with previously characterized serotype 14 and 2 CPSs, N-acetylgalactosamine replaces galactose as the sugar bearing the sialic acid residue in the side chain. Serological analyses of the cross-reaction of serotype 1/2 with serotypes 1 and 2 and that between serotypes 1 and 14 suggested that the side chain, and more particularly the terminal sialic acid, constitutes one important epitope for serotypes 1/2 and 2. The side chain is also an important serological determinant for serotype 1, yet sialic acid seems to play a limited role. In contrast, the side chain does not seem to be part of a major epitope for serotype 14. These results contribute to the understanding of the relationship between S. suis serotypes and provide the basis for improving diagnostic tools.

Revealing the Compact Structure of Lactic Acid Bacterial Heteroexopolysaccharides by SAXS and DLS.

Molecular structures of exopolysaccharides are required to understand their functions and the relationships between the structure and physical and rheological properties. Small-angle X-ray scattering and dynamic light scattering were used in conjunction with molecular modeling to characterize solution structures of three lactic acid bacterial heteroexopolysaccharides (HePS-1, HePS-2, and HePS-3). Values of radius of gyration RG, cross-sectional radius of gyration RXS, approximate length L, and hydrodynamic diameter were not directly proportional to the molar mass and indicated the HePSs adopted a compact coil-like rather than an extended conformation. Constrained molecular modeling of 15000 randomized HePS-1 conformers resulted in five best-fit structures with R factor of 3.9-4.6% revealing random coil-like structure. Φ and Ψ angle analysis of glycosidic linkages in HePS-1 structures suggests Galf residues significantly influence the conformation. Ab initio scattering modeling of HePS-2 and HePS-3 gave excellent curve fittings with χ2 of 0.43 and 0.34 for best-fit models, respectively, compatible with coil-like conformation. The findings disclose solution behavior of HePS relevant for their interactions with biomacromolecules, for example, milk proteins.

Structural characterization of an exopolysaccharide produced by two strains of Lacticaseibacilluscasei.

Exopolysaccharides (EPSs) were isolated and purified from Lacticaseibacillus casei strains type V and RW-3703M grown under various fermentation conditions (carbon source, incubation temperature, and duration). Identical 1H NMR spectra were obtained in all cases. The molar mass determined by size-exclusion chromatography coupled with multi-angle light scattering was different for the two strains and in different culture media. The primary structure was elucidated using chemical and spectroscopic techniques. Monosaccharide and absolute configuration analyses gave the following composition: d-Glc, 1; d-Gal, 2; l-Rha, 2; d-GlcNAc, 1. Methylation analysis indicated the presence of 4-linked Glc, terminal and 6-linked Gal, terminal and 3-linked Rha, and 3,4,6-linked GlcNAc. On the basis of one- and two-dimensional 1H and 13C NMR data, the structure of the EPS was consistent with the following hexasaccharide repeating unit: {4)[Rhap(α1-3)][Galp(α1-6)]GlcpNAc(ß1-6)Galp(α1-3)Rhap(ß1-4)Glcp(ß1-}n. Complete 1H and 13C NMR assignments are reported.

Group B Streptococcus and Streptococcus suis capsular polysaccharides induce chemokine production by dendritic cells via Toll-like receptor 2- and MyD88-dependent and -independent pathways.

Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1ß (IL-1ß), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.

Structure determination of Streptococcus suis serotype 14 capsular polysaccharide.

The capsular polysaccharide (CPS) of Streptococcus suis serotype 14 was purified, chemically modified, and characterized. Sugar and absolute configuration analyses gave the following CPS composition: D-Gal, 3; D-Glc, 1; D-GlcNAc, 1; D-Neu5Ac, 1. The Sambucus nigra lectin, which recognizes the Neu5Ac(α2-6)Gal/GalNAc sequence, showed binding to the native CPS. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. It was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analyses, (1)H and (13)C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [6)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3)]Gal(ß1-3)Gal(ß1-4)Glc(ß1-](n). S. suis serotype 14 CPS has an identical sialic acid-containing side chain as serotype 2 CPS, but differs by the absence of rhamnose in its composition. The same side chain is also present in group B Streptococcus type Ia CPS, except that in the latter sialic acid is 2,3- rather than 2,6-linked to the following galactose. A correlation between the S. suis CPS sequence and genes of the serotype 14 cps locus encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit is proposed.

Streptococcus suis capsular polysaccharide inhibits phagocytosis through destabilization of lipid microdomains and prevents lactosylceramide-dependent recognition.

Streptococcus suis type 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans. S. suis infects the host through the respiratory route, reaches the bloodstream, and persists until breaching into the central nervous system. The capsular polysaccharide (CPS) of S. suis type 2 is considered a key virulence factor of the bacteria. Though CPS allows S. suis to adhere to the membrane of cells of the immune system, it provides protection against phagocytosis. In fact, nonencapsulated mutants are easily internalized and killed by macrophages and dendritic cells. The objective of this work was to study the molecular mechanisms by which the CPS of S. suis prevents phagocytosis. By using latex beads covalently linked with purified CPS, it was shown that CPS itself was sufficient to inhibit entry of both latex beads and bystander fluorescent beads into macrophages. Upon contact with macrophages, encapsulated S. suis was shown to destabilize lipid microdomains at the cell surface, to block nitric oxide (NO) production during infection, and to prevent lactosylceramide accumulation at the phagocytic cup during infection. In contrast, the nonencapsulated mutant was easily internalized via lipid rafts, in a filipin-sensitive manner, leading to lactosylceramide recruitment and strong NO production. This is the first report to identify a role for CPS in lipid microdomain stability and to recognize an interaction between S. suis and lactosylceramide in phagocytes.

Capsular polysaccharide switching in Streptococcus suis modulates host cell interactions and virulence.

The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.

Structure determination of Streptococcus suis serotype 2 capsular polysaccharide.

The capsular polysaccharide (CPS) of Streptococcus suis serotype 2 was isolated, purified, chemically modified, and characterized. Sugar and absolute configuration analyses of the CPS gave the following composition: D-Gal, 3; D-Glc, 1; D-GlcNAc, 1; D-Neu5Ac, 1; L-Rha, 1. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. The CPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analysis, 1H and 13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [4)[Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)]Gal(beta1-4)[Gal(alpha1-3)]Rha(beta1-4)Glc(beta1-]n. The backbone sequence was found to be identical to that of Streptococcus agalactiae or group B Streptococcus (GBS) type VIII and Streptococcus pneumoniae type 23F. The S. suis CPS shares the sequence Neu5Ac-Gal-GlcNAc-Gal in common with GBS types Ia, Ib, II, III, and IV CPSs but differs from them by the presence of rhamnose and the fact that sialic acid is 2,6- rather than 2,3-linked to the following Gal. A correlation between the S. suis CPS sequence and genes of the serotype 2 cps locus encoding putative enzymes responsible for the biosynthesis of the repeating unit was tentatively established.

The exopolysaccharide properties and structures database: EPS-DB. Application to bacterial exopolysaccharides.

The EPS Database (EPS-DB) is a web-based, platform-independent database of bacterial exopolysaccharides (EPSs) providing access to detailed structural, taxonomic, growth conditions, functional properties, genetic, and bibliographic information for EPSs. It is freely available on the Internet as a website at http://www.epsdatabase.com. Several structural data representation schemes are used following the most commonly accepted formats. This guarantees full interoperability with other structural, experimental, and functional databases in the area of glycoscience. The scientific usage of EPS-DB throughout a user-friendly interface is presented with a subsection of the database exemplified by EPSs from lactic acid bacteria.

Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus.

The capsular polysaccharide (CPS) represents a key virulence factor for most encapsulated streptococci. Streptococcus suis and Group B Streptococcus (GBS) are both well-encapsulated pathogens of clinical importance in veterinary and/or human medicine and responsible for invasive systemic diseases. S. suis and GBS are the only Gram-positive bacteria which express a sialylated CPS at their surface. An important difference between these two sialylated CPSs is the linkage between the side-chain terminal galactose and sialic acid, being α-2,6 for S. suis but α-2,3 for GBS. It is still unclear how sialic acid may affect CPS production and, consequently, the pathogenesis of the disease caused by these two bacterial pathogens. Here, we investigated the role of sialic acid and the putative effect of sialic acid linkage modification in CPS synthesis using inter-species allelic exchange mutagenesis. To this aim, a new molecular biogenetic approach to express CPS with modified sialic acid linkage was developed. We showed that sialic acid (and its α-2,6 linkage) is crucial for S. suis CPS synthesis, whereas for GBS, CPS synthesis may occur in presence of an α-2,6 sialyltransferase or in absence of sialic acid moiety. To evaluate the effect of the CPS composition/structure on sialyltransferase activity, two distinct capsular serotypes within each bacterial species were compared (S. suis serotypes 2 and 14 and GBS serotypes III and V). It was demonstrated that the observed differences in sialyltransferase activity and specificity between S. suis and GBS were serotype unrestricted. This is the first time that a study investigates the interspecies exchange of capsular sialyltransferase genes in Gram-positive bacteria. The obtained mutants represent novel tools that could be used to further investigate the immunomodulatory properties of sialylated CPSs. Finally, in spite of common CPS structural characteristics and similarities in the cps loci, sialic acid exerts differential control of CPS expression by S. suis and GBS.

Interaction between structurally different heteroexopolysaccharides and ß-lactoglobulin studied by solution scattering and analytical ultracentrifugation.

Despite a very large number of bacterial exopolysaccharides have been reported, detailed knowledge on their molecular structures and associative interactions with proteins is lacking. Small-angle X-ray scattering, dynamic light scattering and analytical ultracentrifugation (AUC) were used to characterize the interactions of six lactic acid bacterial heteroexopolysaccharides (HePS-1-HePS-6) with ß-lactoglobulin (BLG). Compared to free HePSs, a large increase in the X-ray radius of gyration RG, maximum length L and hydrodynamic diameter dH of HePS-1-HePS-4 mixed with BLG revealed strong aggregation, the extent of which depended on the compact conformation and degree of branching of these HePSs. No significant effects were observed with HePS-5 and HePS-6. Turbidity and AUC analyses showed that both soluble and insoluble BLG-HePS complexes were formed. The findings provide new insights into the role of molecular structures in associative interactions between HePSs and BLG which has relevance for various industrial applications.

Effect of repeat unit structure and molecular mass of lactic acid bacteria hetero-exopolysaccharides on binding to milk proteins.

Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero-exopolysaccharides (HePSs) of 0.14-4.9MDa from lactic acid bacteria to different milk proteins (ß-casein, κ-casein, native and heat-treated ß-lactoglobulin) at pH 4.0-5.0. Maximum binding capacity (RUmax) and apparent affinity (KA,app) were HePS- and protein-dependent and varied for example 10- and 600-fold, respectively, in the complexation with native ß-lactoglobulin at pH 4.0. Highest RUmax and KA,app were obtained with heat-treated ß-lactoglobulin and ß-casein, respectively. Overall, RUmax and KA,app decreased 6- and 20-fold, respectively, with increasing pH from 4.0 to 5.0. KA,app was influenced by ionic strength and temperature, indicating that polar interactions stabilize HePS-protein complexes. HePS size as well as oligosaccharide repeat structure, conferring chain flexibility and hydrogen bonding potential, influence the KA,app.

A single amino acid polymorphism in the glycosyltransferase CpsK defines four Streptococcus suis serotypes.

The capsular polysaccharide (CPS) is the major virulence factor of the emerging zoonotic pathogen Streptococcus suis. CPS differences are also the basis for serological differentiation of the species into 29 serotypes. Serotypes 2 and 1/2, which possess identical gene content in their cps loci, express CPSs that differ only by substitution of galactose (Gal) by N-acetylgalactosamine (GalNAc) in the CPS side chain. The same sugar substitution differentiates the CPS of serotypes 14 and 1, whose cps loci are also identical in gene content. Here, using mutagenesis, CPS structural analysis, and protein structure modeling, we report that a single amino acid polymorphism in the glycosyltransferase CpsK defines the enzyme substrate predilection for Gal or GalNAc and therefore determines CPS composition, structure, and strain serotype. We also show that the different CPS structures have similar antiphagocytic properties and that serotype switching has limited impact on the virulence of S. suis.

Structure determination of the neutral exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1.

The neutral exopolysaccharide (NPS) of Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1 was purified and characterized. The molecular mass was 5.0×10(6) g/mol. Sugar and absolute configuration analyses gave the following composition: d-Glc, 1; d-Gal, 1.5. The NPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analyses, (1)H and (13)C nuclear magnetic resonance, and mass spectrometry of the NPS or of its specifically modified products allowed determining the repeating unit sequence: {2)Glc(α1-3)Glc(ß1-3)[Gal(ß1-4)]Gal(ß1-4)Gal(α1-}n. The structure is compared to that of exopolysaccharides produced by other Lactobacillus bulgaricus strains.

Sialylation of Streptococcus suis serotype 2 is essential for capsule expression but is not responsible for the main capsular epitope.

The capsular polysaccharide is a critical virulence factor of the swine and zoonotic pathogen Streptococcus suis serotype 2. The capsule of this bacterium is composed of five different sugars, including terminal sialic acid. To evaluate the role of sialic acid in the pathogenesis of the infection, the neuC gene, encoding for an enzyme essential for sialic acid biosynthesis, was inactivated in a highly virulent S. suis serotype 2 strain. Using transmission electron microscopy, it was shown that inactivation of neuC resulted in loss of expression of the whole capsule. Compared to the parent strain, the ΔneuC mutant strain was more phagocytosed by macrophages and was also severely impaired in virulence in a mouse infection model. Both native and desialylated S. suis serotype 2 purified capsular polysaccharides were recognized by a polyclonal anti-whole cell S. suis serotype 2 serum and a monospecific polyclonal anti-capsule serotype 2 serum. In contrast, only the native capsular polysaccharide was recognized by a monoclonal antibody specific for the sialic acid moiety of the serotype 2 capsule. Together, our results infer that sialylation of S. suis serotype 2 may be essential for capsule expression, but that this sugar is not the main epitope of this serotype.

Synthesis and characterization of new permanently charged poly(amidoammonium) salts and evaluation of their DNA complexes for gene transport.

A new series of linear and permanently charged poly(amidoammonium) salts were synthesized in order to investigate the influence of their ionic and hydrophobic contents on both the cytotoxicity and the transfection mediated by polycation-DNA complexes. The poly(amidoammonium) salts were prepared by chemical modification of a parent poly(amidoamine) containing two tertiary amino groups per structural unit: one incorporated into the main chain and the other fixed at the end of a short bismethylene spacer. The permanent charges were introduced through a quaternization reaction involving iodomethane or 1-iodododecane as an alkylating agent. Under appropriate conditions, the methylation reaction was found to be regioselective, allowing the quaternization of either the side chains or both the side chains and the backbone. Under physiological salt conditions (150 mM NaCl), all of the poly(amidoammonium) salts self-assembled with DNA to form complexes. High proportions of highly quaternized polycation provided better defined morphology to the polycation-DNA complexes. Complexes formed from unquaternized polycation were less cytotoxic than branched poly(ethyleneimine) (25 kDa). At high polycation-DNA weight ratios, the introduction of permanent charges generated a significant increase in the cytotoxicity, but no patent correlation could be established with the amount and the position of the permanent charges. Only complexes formed from polycations with quaternized backbone were able to generate significant gene expression, which was putatively attributed to a better defined toroidal-like morphology together with a higher stability, as suggested by zeta potential measurements. The incorporation of dodecane side chains on highly charged polycations severely amplified the cytotoxicity so that, in return, the transfection level was dramatically affected.

Effect of exopolysaccharides on phage-host interactions in Lactococcus lactis.

In this study, we report that Lactococcus lactis strains producing exopolysaccharides (EPS) are sensitive to virulent phages. Eight distinct lytic phages (Q61 to Q68) specifically infecting Eps(+) strains were isolated in 47 buttermilk samples obtained from 13 North American factories. The eight phages were classified within the 936 species by the multiplex PCR method, indicating that these phages are not fundamentally distinct from those infecting Eps(-) L. lactis strains. The host range of these phages was determined with 19 Lactococcus strains, including 7 Eps(+) and 12 Eps(-) cultures. Three phages (Q62, Q63, and Q64) attacked only the Eps(+) strain SMQ-419, whereas the five other phages (Q61, Q65, Q66, Q67, and Q68) infected only the Eps(+) strain SMQ-420. The five other Eps(+) strains (H414, MLT2, MLT3, SMQ-461, and SMQ-575) as well as the 12 Eps(-) strains were insensitive to these phages. The monosaccharide composition of the polymer produced by the seven Eps(+) strains was determined. The EPS produced by strains MLT3, SMQ-419, and SMQ-575 contained glucose, galactose, and rhamnose. The EPS fabricated by H414 contained only galactose. The EPS made by MLT2, SMQ-420, and SMQ-461 contained glucose and galactose. These findings indicate that the sugar composition of the EPS has no effect on phage sensitivity. The plasmid encoding the eps operon was cured from the two phage-sensitive strains. The cured derivatives were still phage sensitive, which indicates that EPS are not necessary for phage infection. Phage adsorption assays showed that the production of EPS does not confer a significant phage resistance phenotype.

Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595M and R.

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillus rhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical (1)H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; D-glucose, 2; D-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional (1)H and (13)C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: [3Rha alpha-3Glc beta-3[Gal4,6(R)Py alpha-2]Rha alpha-3Rha alpha-3Rha alpha-2Glc alpha-](n) where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete (1)H and (13)C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group.

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.