Capsular Polymer Research Articles

Capsules and Extracellular Polysaccharides in Escherichia coli and Salmonella.

Escherichia coli and Salmonella isolates produce a range of different polysaccharide structures that play important roles in their biology. E. coli isolates often possess capsular polysaccharides (K antigens), which form a surface structural layer. These possess a wide range of repeat-unit structures. In contrast, only one capsular polymer (Vi antigen) is found in Salmonella, and it is confined to typhoidal serovars. In both genera, capsules are vital virulence determinants and are associated with the avoidance of host immune defenses. Some isolates of these species also produce a largely secreted exopolysaccharide called colanic acid as part of their complex Rcs-regulated phenotypes, but the precise function of this polysaccharide in microbial cell biology is not fully understood. E. coli isolates produce two additional secreted polysaccharides, bacterial cellulose and poly-N-acetylglucosamine, which play important roles in biofilm formation. Cellulose is also produced by Salmonella isolates, but the genes for poly-N-acetylglucosamine synthesis appear to have been lost during its evolution toward enhanced virulence. Here, we discuss the structures, functions, relationships, and sophisticated assembly mechanisms for these important biopolymers.

Membrane Vesicles: A Common Feature in the Extracellular Matter of Cold-Adapted Antarctic Bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22(T), Shewanella vesiculosa M7(T), Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23(T), and Marinobacter guineae M3B(T)) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22(T), the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22(T) obtained at 4 and 18 degrees C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.

Structure of the high-molecular weight exopolysaccharide isolated from Lactobacillus pentosus LPS26

The strain Lactobacillus pentosus LPS26 produces a capsular polymer composed of a high- (2.0 × 10 6 Da) (EPS A) and a low-molecular mass (2.4 × 10 4 Da) (EPS B) polysaccharide when grown on semi-defined medium containing glucose as the carbon source. The structure of EPS A and its deacetylated form has been determined by monosaccharide and methylation analysis as well as by 1D/2D NMR studies ( 1H and 13C). We conclude that EPS A is a charged heteropolymer, with a composition of d-glucose, d-glucuronic acid and l-rhamnose in a molar ratio 1:2:2. The repeating unit is a pentasaccharide with two O-acetyl groups at O-4 of the 3-substituted α- d-glucuronic acid and at O-2 of the 3-substituted β- l-rhamnose, respectively. →4)-α- d-Glc p-(1→3)-α- d-Glc pA4Ac-(1→3)-α- l-Rha p-(1→4)-α- d-Glc pA-(1→3)-β- l-Rha p2Ac-(1→ This unbranched structure is not common in EPSs produced by Lactobacilli. Moreover, the presence of acetyl groups in the structure is an unusual feature which has only been reported in L. sake 0–1 [Robijn et al. Carbohydr. Res., 1995, 276, 117–136].

Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases

The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in α-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. Subsequent structure–function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure–function relations and for phylogenetic sorting of these important enzymes.

Culture Conditions Determine the Balance between Two Different Exopolysaccharides Produced by Lactobacillus pentosus LPS26

Lactobacillus pentosus LPS26, isolated from a natural fermentation of green olives, produces a capsular polymer constituted of two exopolysaccharides (EPS): EPS A, a high-molecular-weight (high-Mw) polysaccharide (1.9x10(6) Da) composed of glucose and rhamnose (3:1), and EPS B, a low-Mw polysaccharide (3.3x10(4) Da) composed of glucose and mannose (3:1). Fermentation experiments in a chemically semidefined medium with different carbon sources (glucose, fructose, mannitol, and lactose) showed that all of them except fructose supported EPS A production rather than EPS B production. The influence of temperature and pH was further analyzed. As the temperature dropped, increased synthesis of both EPS was detected. The control of pH especially enhanced EPS B production. With regard to this, the maximum total EPS production (514 mg liter-1) was achieved at a suboptimal growth temperature (20 degrees C) and pH 6.0. Continuous cultures showed that EPS A, synthesized mainly at low dilution rates, is clearly dependent on the growth rate, whereas EPS B synthesis was hardly affected. EPS production was also detected in supplemented skimmed milk, but no increase on the viscosity of the fermented milk was recorded. This could be linked to the high proportion of the low-Mw polysaccharide produced in these conditions in contrast to that observed in culture media. Overall, the present study shows that culture conditions have a clear impact on the type and concentration of EPS produced by strain LPS26, and consequently, these conditions should be carefully selected for optimization and application studies. Finally, it should be noted that this is, to our knowledge, the first report on EPS production by L. pentosus.

Characterization of outer membrane vesicles released by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3

Pseudoalteromonas antarctica NF3 is an Antarctic psychrotolerant Gram-negative bacterium that accumulates large amounts of an extracellular polymeric substance (EPS) with high protein content. Transmission electron microscopy analysis after high-pressure freezing and freeze substitution (HPF-FS) shows that the EPS is composed of a capsular polymer and large numbers of outer membrane vesicles (OMVs). These vesicles are bilayered structures and predominantly spherical in shape, with an average diameter of 25-70 nm, which is similar to what has been observed in OMVs from other Gram-negative bacteria. Analyses of lipopolysaccharide (LPS), phospholipids and protein profiles of OMVs are consistent with the bacterial outer membrane origin of these vesicles. In an initial attempt to elucidate the functions of OMVs proteins, we conducted a proteomic analysis on 1D SDS-PAGE bands. Those proteins putatively identified match with outer membrane proteins and proteins related to nutrient processing and transport in Gram-negative bacteria. This approach suggests that OMVs present in the EPS from P. antarctica NF3, might function to deliver proteins to the external media, and therefore play an important role in the survival of the bacterium in the extreme Antarctic environment.

Ultrastructural Analysis of the Extracellular Matter Secreted by the Psychrotolerant Bacterium Pseudoalteromonas antarctica NF3

The psychrotolerant strain Pseudoalteromonas antarctica NF3, a Gram-negative bacterium isolated from muddy soil samples of Antarctica, secretes large amounts of a mucoid exopolymer with a high protein content. It has self-assembly properties and capacity to coat and protect liposomes against surfactants. We examined the ultrastructure of P. antarctica and the extracellular matter it secretes by transmission electron microscopy (TEM) after high-pressure freezing, freeze substitution (HPF-FS), and Epon embedding, and compared this with information obtained by conventional methods. The improvements brought about by HPF-FS to the ultrastructural preservation of the extracellular matter allowed us to establish for the first time, in P. antarctica NF3, the presence of two components: a large amount of cell-derived outer membrane vesicles containing proteins and a capsular polymer around the cells.

Analysis of the G93E mutant allele of KpsM, the membrane component of an ABC transporter involved in polysialic acid translocation in Escherichia coli K1

KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1. The kpsMG93E allele is a point mutation in the first cytoplasmic loop (Cl) of KpsM which partially disrupts translocation of the capsule. While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface.

Translocation and Surface Expression of Lipidated Serogroup B Capsular Polysaccharide in Neisseria meningitidis

The capsule of N. meningitidis serogroup B, (alpha2-->8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3' end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.

Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30: STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE OUTER MEMBRANE LIPOPROTEIN Wza

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. WzaHis6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of WzaHis6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the WzaHis6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.

Bioactive capsular polysaccharide from the thermophilic Mastigocladus laminosus (Cyanophyceae/Cyanobacteria): demonstration of anti-inflammatory properties

The anti-inflammatory, antimicrobial and cytotoxic activities of the capsular polymer produced by the thermophilic blue-green alga Mastigocladus laminosus were tested. Used at concentrations ranging fom 10 -5 to 1 mg ml -1 , the extracts didn't show an inhibition of human cell activity or a significant effect on the growth of Gram-negative and Gram-positive bacteria, and of a yeast strain. However, a very significant and dose-dependent anti-inflammatory effect was obtained when the extracts were topically applied on croton oil-induced oedema in mice ear skin. The application of a partial hydrolysate of the polymer only formed by oligosaccharides with degrees of polymerisation ranging from 2 to 5 reduced the oedema in a similar way as the polymer, indicating that the anti-inflammatory activity is associated with the polysaccharidic fraction.

Identification and Molecular Cloning of a Heparosan Synthase fromPasteurella multocida Type D

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.

Identification of two glycosylated components of Mycoplasma penetrans: a surface-exposed capsular polysaccharide and a glycolipid fraction.

Among the wall-less mycoplasmas only a few species have been identified with a capsule at their cell surface. Mycoplasma penetrans is a recently identified mycoplasma with unique morphology, isolated from HIV-infected patients. Using transmission electron microscopy, it was found that M. penetrans is surrounded by capsular material 11 nm (strain GTU-54-6A1) to 30 nm (strain HF-2) thick, which can be stained with ruthenium red and labelled with cationized ferritin. The polysaccharide composition of this capsule was indicated by its staining with periodic acid-thiocarbohydrazide silver proteinate and the abolition of ruthenium red staining of the cell surface by neuraminidase treatment. In addition, proteinase K treatment of the M. penetrans cells resulted in removal of the capsule, suggesting that polypeptides may contribute in anchoring it to the membrane or in its stability. Two different types of glycosylated material were detected in mycoplasma extracts by SDS-PAGE and periodic acid-Schiff staining. The first component was a high-molecular-mass material, which was heat- and proteinase-K-labile and which probably constitutes the capsular polymer. The other component was a low-molecular-mass glycolipid fraction, which was proteinase-K-, heat- and EDTA-resistant. The identification of a capsule at the M. penetrans cell surface is of particular interest for a mycoplasma which has been shown to adhere to various host cells and to penetrate into their intracellular compartments. The capsule may have significance in the pathogenesis of disease associated with infection by this organism.

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. When the bacterium was grown at 37 degrees C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 microg/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40 degrees no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of Nacetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 microM.

Analysis of the G93E mutant allele of KpsM, the membrane component of an ABC transporter involved in polysialic acid translocation in Escherichia coli K1

KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1. The kpsMG93E allele is a point mutation in the first cytoplasmic loop (CI) of KpsM which partially disrupts translocation of the capsule. While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface.

Metal accumulation by immobilized cyanobacterial mats from a thermal spring

Abstract The metal‐binding properties of lyophilized cyanobacterial mats occurring in a thermal spring were investigated. The general binding efficiency decreased in the order Cu > Ni > Pb > Cr. Most metal sorption occurred within 15 min, and it was strongly pH‐dependent. The sorption of Cu was depressed in the case of competition with other metals or cations. The metal‐binding properties are probably due to a high density of anionic charges, especially carboxyls, identified in the capsular polymer. The feasability to use columns packed with the biomass immobilized with silica gel for the removal of metals is demonstrated. The metals were successfully desorbed and these columns were effective during several adsorption‐desorption cycles.

Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis

Bacillus anthracis produces a gamma-linked poly-D-glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low-molecular-weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region alone caused production of a high-molecular-weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51,460. By in vitro transcription-translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian gamma-glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly-D-glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low-molecular-weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.

Determination of the structure of the capsular antigen of Escherichia coli O8:K46:H30, using FABMS and 2D-NMR spectroscopy

The structure of the capsular antigen from Escherichia coli O8:K46:H30 was elucidated by methylation analysis and 1D and 2D 1H- and 13C-NMR spectroscopy, and by methylation analysis, 1D- and 2D-NMR spectroscopy, and FABMS of the oligosaccharide-alditol obtained after dephosphorylation of the polymer with aqueous hydrofluoric acid. The capsular polymer is of the teichoic acid type and has the following repeating unit. ▪

Structure of the capsular polymer of Actinobacillus pleuropneumoniae serotype 6

The capsular polymer of Actinobacillus pleuropneumoniae serotype 6 (ATCC 33590) was investigated by methylation, dephosphorylation, partial hydrolysis, and 1H- and 13C-n.m.r. methods. It was found to be a high-molecular-weight polymer composed of triglycosylglycerol phosphate repeating-units joined through monophosphate diester linkages, and has the structure: ▪

Purification and partial characterization of the capsular polymer of Haemophilus pleuropneumoniae serotype 5.

The capsular polymer (CP) of Haemophilus pleuropneumoniae serotype 5 was purified, and its chemical composition was analyzed. Radioimmunoassay experiments showed that the maximum amount of CP could be obtained from broth cultures of bacteria in the late stationary phase, rather than from bacteria washed off agar plates. The CP was precipitated from culture supernatant with 5 mM hexadecyltrimethylammonium bromide (Cetavlon) and solubilized with 0.4 M NaCl. Ninety percent of the CP in the culture supernatant was precipitated with Cetavlon, although some material remained insoluble after NaCl extraction. The CP was further purified by phenol extraction, ultracentrifugation, and Sepharose CL-4B gel filtration. The Kav of the CP from Sepharose CL-4B chromatography was 0.33. The CP preparation contained 85% hexosamine, 12% hexose, 3% phosphate, 0.17% protein, 0.20% nucleic acid, and 0.01% endotoxin. Thin-layer chromatography, an amino acid analyzer, and a glucose oxidase colorimetric kit were used to identify the sugar components of the hydrolyzed CP as glucosamine and glucose. Analysis of the native CP by 13C nuclear magnetic resonance indicated that amino, N-acetyl, and carboxyl groups were present and that the CP was a disaccharide.