Bacteroides fragilis synthesizes a DNA invertase affecting both a local and a distant region

The activity of a fourth conserved tyrosine site-specific recombinase (Tsr) of Bacteroides fragilis was characterized. Its gene, tsr19, is adjacent to mpi, encoding the global DNA invertase regulating capsular polysaccharide biosynthesis. Unlike the other described Tsrs of B. fragilis, Tsr19 brings about inversion of two DNA regions, one local and one located distantly.     

Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.     

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.     

Tyrosine site-specific recombinases mediate DNA inversions affecting the expression of outer surface proteins of Bacteroides fragilis

The chromosome of Bacteroides fragilis has been shown to undergo 13 distinct DNA inversions affecting the expression of capsular polysaccharides and mediated by a serine site-specific recombinase designated Mpi. In this study, we demonstrate that members of the tyrosine site-specific recombinase family, conserved in B. fragilis, mediate additional DNA inversions of the B. fragilis genome. These DNA invertases flip promoter regions in their immediate downstream region. The genetic organization of the genes regulated by these invertible promoter regions suggests that they are operons and many of the products are predicted to be outer membrane proteins. Phenotypic analysis of a deletion mutant of one of these DNA invertases, tsr15 (aapI), which resulted in the promoter region for the downstream genes being locked ON, confirmed the synthesis of multiple surface proteins by this operon. In addition, this deletion mutant demonstrated an autoaggregative phenotype and showed significantly greater adherence than wild-type organisms in a biofilm assay, suggesting a possible functional role for these phase-variable outer surface proteins. This study demonstrates that DNA inversion is a universal mechanism used by this commensal microorganism to phase vary expression of its surface molecules and involves at least three conserved DNA invertases from two evolutionarily distinct families.     

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.     

Capsular polysaccharides and lipopolysaccharides from two Bacteroides fragilis reference strains: chemical and immunochemical characterization.

Fermentor growth of Bacteroides fragilis under controlled conditions in a complex medium containing 1% glucose and 10% fetal calf serum resulted in high yields of bacteria. After hot phenol-water extraction of the organisms, capsular polysaccharide was isolated from the aqueous phase and purified by Sephacryl S-300 chromatography in a buffer with 3% sodium deoxycholate. Lipopolysaccharide was isolated by phenol-chloroform-light petroleum ether extraction. The capsular polysaccharide from B. fragilis strain NCTC 9343 contained six sugars: L-fucose, D-galactose, D- and L-quinovosamine, D-glucosamine, and galacturonic acid. The capsule of strain ATCC 23745 also contained D-glucose, L-fucosamine, L-rhamnosamine, and a 3-amino-3,6-dideoxyhexose but lacked D-quinovosamine. The latter capsule also contained alanine (4%). The capsular polysaccharides were different immunochemically by ELISA inhibition. The lipopolysaccharide of both strains contained the same sugars (L-rhamnose, D-glucose, D-galactose, and D-glucosamine) and fatty acids (13-methyl-tetradecanoic and 3-hydroxy-hexadecanoic and 3-hydroxy-15 methyl-hexadecanoic as major constituents) and were identical by ELISA inhibition.     

Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra

Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum, RBL5808 and RBL5812, revealed independent Tn5 transposon integrations in a single gene, designated exo5. As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.     

DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.     

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.     

细菌荚膜多糖的化学结构

荚膜是一些细菌所具有的表层结构,与多种疾病有着密切联系。细菌荚膜多糖不仅结构复杂,而且在免疫活性方面发挥着重要的作用。同一种细菌根据其荚膜多糖的抗原性不同可分为不同的血清型,不同血清型细菌荚膜多糖的化学结构也存在差异。以荚膜多糖为基础的疫苗正在积极研究开发当中,对不同致病细菌荚膜多糖具体化学结构的掌握是疫苗得到许可的必备条件之一。本文对致病细菌荚膜多糖的化学结构进行了归纳和总结,以期为荚膜多糖的化学结构研究和疫苗开发提供参考。     

Polysaccharides production by Rhizobium phaseoli and the typing of their excreted anionic polysaccharides

Abstract The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity. Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-β-hydroxybutyric acid (PHB) and/or glycogen in their cells. The latter can then be used as C-sources for further synthesis of EPS and CPS. Some strains are only very poor producers or do not produce at all. Nine strains of R. phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R. leguminosarum (6 strains) and R. trifolli (9 strains). Three strains of R. phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.     

Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.     

Selective synthesis of polysaccharides by Rhizobium trifolii, strain TA-1

Abstract Rhizobium trifolii strain TA-1, produces one of each of the exocellular polysaccharides EPS, CPS and β-1,2-glucans as a major product during cultivation in glutamic acid-mannitol-salts (GMS) medium at 25°. In batch culture, the major exocellular polysaccharide product was acidic exopolysaccharide (EPS) under conditions of air saturation; capsular polysaccharide (CPS) under conditions of N-limitation and moderate oxygen supply; and cyclic β-1,2-glucans at high cell density and severe oxygen limitation.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

淫羊藿多糖的分离纯化及结构初步分析

从淫羊藿中提取多糖并鉴定其初步结构和单糖组成.采用超声-水提醇沉法提取粗多糖、Sevag法去蛋白、DEAE-52纤维素及Sephadex G-100柱层析法纯化得到淫羊藿多糖EPSⅠ-1和EPSⅡ-1.应用紫外光谱法和红外光谱法对其结构做初步分析.采用高效液相色谱法(HPLC)测定其单糖组成及摩尔比.均一的EPSⅠ-1和EPSⅡ-1多糖在紫外和红外中具有多糖的特征吸收峰,组成中含有吡喃环结构;EPSⅠ-1的单糖组成为鼠李糖和葡萄糖,摩尔比为1:1.13;EPSⅡ-1的单糖组成为果糖、葡萄糖和一个不确定的糖,摩尔比为1:1.91.有效地分离纯化了淫羊藿多糖,这为淫羊藿多糖的广泛应用奠定了实验基础.     

Chemical composition and bioactivities of a water-soluble polysaccharide from the endodermis of shaddock

The chemical composition of shaddock (Citrus paradisi) mainly consisted of polyphenols, proteins and polysaccharides. However, polysaccharides from shaddock materials have received much less consideration than polyphenols. Herein, a water-soluble neutral polysaccharide from the endodermis of shaddock was isolated and showed good bioactivities. Crude polysaccharides from the endodermis of shaddock (EPS) was extracted with hot water and separated on a DEAE Sepharose FF gel filtration column to obtain NEPS. The IR and UV spectra of NEPS showed that NEPS was mainly composed of polysaccharide and there are no proteins existing in NEPS. The DPPH radical scavenging and reducing power of NEPS are much lower than those of crude EPS; however, Citrus flavonoids significantly improved the DPPH radical scavenging potential and reducing power of NEPS. The crude EPS (5mg/mL) showed a similar inhibitory effect (77.92±5.03%) with NEPS (5mg/mL) (74.63±4.71%) on α-amylase.     

The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell

The export of large negatively charged capsular polysaccharides across the outer membrane represents a significant challenge to Gram negative bacteria. In the case of Escherichia coli group 2 capsular polysaccharides, the mechanism of export across the outer membrane was unknown, with no identified candidate outer membrane proteins. In this paper we demonstrate that the KpsD protein, previously believed to be a periplasmic protein, is an outer membrane protein involved in the export of group 2 capsular polysaccharides across the outer membrane. We demonstrate that KpsD and KpsE are located at the poles of the cell and that polysaccharide biosynthesis and export occurs at these polar sites. By in vivo chemical cross-linking and MALDI-TOF-MS analysis we demonstrate the presence of a multi-protein biosynthetic/export complex in which cytoplasmic proteins involved in polysaccharide biosynthesis could be cross-linked to proteins involved in export across the inner and outer membranes. In addition, we show that the RhsA protein, of previously unknown function, could be cross-linked to the complex and that a rhsA mutation reduces K5 biosynthesis suggesting a role for RhsA in coupling biosynthesis and export.     

革兰氏阳性菌荚膜多糖研究进展*

细菌的荚膜多糖是生物膜的重要组成部分,在细菌的生长分裂、维持细胞壁形态、抵御外界环境以及免疫反应等方面都起到重要作用。在致病菌中,荚膜多糖常作为一种毒力因子发挥作用。在革兰氏阳性菌中,荚膜多糖的化学结构、生物合成过程及功能应用越来越受到关注。讨论了革兰氏阳性菌中部分致病菌的荚膜多糖与非致病菌表面多糖的分布位置、化学组成及其结构特异性。重点讨论三种具有代表性的革兰氏阳性致病菌及非致病菌株:肺炎链球菌(Streptococcus pneumonia)、金黄色葡萄球菌(Staphylococcus aureus)及乳酸乳球菌(Lactococcus lactis)。综述革兰氏阳性菌中荚膜多糖生物合成的三种方式:Wzx/Wzy-依赖通路、ABC转运蛋白(ABC transporter)途径及合酶依赖途径,并举例解释了相应多糖的合成过程及相关基因。介绍了革兰氏阳性菌荚膜多糖及表面多糖的生理功能,如屏障保护功能、胞间黏附功能以及参与宿主细胞的免疫反应等。结合荚膜多糖的生物学功能,概述其当前主要研究进展,如构建高耐受工程菌疫苗研制等。结合细菌荚膜多糖的特征差异,对其在医药与工业生产领域的广阔前景提出展望和建议。     

Dual Roles of Capsular Extracellular Polymeric Substances in Photocatalytic Inactivation of Escherichia coli: Comparison of E. coli BW25113 and Isogenic Mutants

The dual roles of capsular extracellular polymeric substances (EPS) in the photocatalytic inactivation of bacteria were demonstrated in a TiO₂-UVA system, by comparing wild-type Escherichia coli strain BW25113 and isogenic mutants with upregulated and downregulated production of capsular EPS. In a partition system in which direct contact between bacterial cells and TiO₂ particles was inhibited, an increase in the amount of EPS was associated with increased bacterial resistance to photocatalytic inactivation. In contrast, when bacterial cells were in direct contact with TiO₂ particles, an increase in the amount of capsular EPS decreased cell viability during photocatalytic treatment. Taken together, these results suggest that although capsular EPS can protect bacterial cells by consuming photogenerated reactive species, it also facilitates photocatalytic inactivation of bacteria by promoting the adhesion of TiO₂ particles to the cell surface. Fluorescence microscopy and scanning electron microscopy analyses further confirmed that high capsular EPS density led to more TiO₂ particles attaching to cells and forming bacterium-TiO₂ aggregates. Calculations of interaction energy, represented by extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) potential, suggested that the presence of capsular EPS enhances the attachment of TiO₂ particles to bacterial cells via acid-base interactions. Consideration of these mechanisms is critical for understanding bacterium-nanoparticle interactions and the photocatalytic inactivation of bacteria.