Glycosyl Composition Research Articles

Water stress and cell wall polysaccharides in the apical root zone of wheat cultivars varying in drought tolerance

Glycosyl composition and linkage analysis of cell wall polysaccharides were examined in apical root zones excised from water-stressed and unstressed wheat seedlings (Triticum durum Desf.) cv. Capeiti ("drought-tolerant") and cv. Creso ("drought sensitive"). Wall polysaccharides were sequentially solubilized to obtain three fractions: CDTA+Na(2)CO(3) extract, KOH extract and the insoluble residue (alpha-cellulose). A comparison between the two genotypes showed only small variations in the percentages of matrix polysaccharides (CDTA+Na(2)CO(3) plus KOH extract) and of the insoluble residues (alpha-cellulose) in water-stressed and unstressed conditions. Xylosyl, glucosyl and arabinosyl residues represented more than 90 mol% of the matrix polysaccharides. The linkage analysis of matrix polysaccharides showed high levels of xyloglucans (23-39 mol%), and arabinoxylans (38-48 mol%) and a low amount of pectins and (1-->3), (1-->4)-beta-D-glucans. The high level of xyloglucans was supported by the release of the diagnostic disaccharide isoprimeverose after Driselase digestion of KOH-extracted polysaccharides. In the "drought-tolerant" cv. Capeiti the mol% of side chains of rhamnogalacturonan I and II significantly increased in response to water stress, whereas in cv. Creso, this increase did not occur. The results support a role of the pectic side chains during water stress response in a drought-tolerant wheat cultivar.

Cryptococcus neoformans Capsular Polysaccharide and Exopolysaccharide Fractions Manifest Physical, Chemical, and Antigenic Differences

The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide (PS) capsule and releases copious amounts of PS into cultures and infected tissues. The capsular PS is a major virulence factor that can elicit protective antibody responses. PS recovered from culture supernatants has historically provided an ample and convenient source of material for structural and immunological studies. Two major assumptions in such studies are that the structural features of the exopolysaccharide material faithfully mirror those of capsular PS and that the isolation methods do not change PS properties. However, a comparison of exopolysaccharide made by two isolation techniques with capsular PS stripped from cells with gamma radiation or dimethyl sulfoxide revealed significant differences in glycosyl composition, mass, size, charge, viscosity, circular-dichroism spectra, and reactivity with monoclonal antibodies. Our results strongly suggest that exopolysaccharides and capsular PS are structurally different. A noteworthy finding was that PS made by cetyltrimethylammonium bromide precipitation had a larger mass and a different conformation than PS isolated by concentration and filtration, suggesting that the method most commonly used to purify glucuronoxylomannan alters the PS. Hence, the method used to isolate PS can significantly influence the structural and antigenic properties of the product. Our findings have important implications for current views of the relationship between capsular PS and exopolysaccharides, for the generation of PS preparations suitable for immunological studies, and for the formulation of PS-based vaccines for the prevention of cryptococcosis.

Cell Wall Carbohydrate Compositions of Strains from the Bacillus cereus Group of Species Correlate with Phylogenetic Relatedness

Members of the Bacillus cereus group contain cell wall carbohydrates that vary in their glycosyl compositions. Recent multilocus sequence typing (MLST) refined the relatedness of B. cereus group members by separating them into clades and lineages. Based on MLST, we selected several B. anthracis, B. cereus, and B. thuringiensis strains and compared their cell wall carbohydrates. The cell walls of different B. anthracis strains (clade 1/Anthracis) were composed of glucose (Glc), galactose (Gal), N-acetyl mannosamine (ManNAc), and N-acetylglucosamine (GlcNAc). In contrast, the cell walls from clade 2 strains (B. cereus type strain ATCC 14579 and B. thuringiensis strains) lacked Gal and contained N-acetylgalactosamine (GalNAc). The B. cereus clade 1 strains had cell walls that were similar in composition to B. anthracis in that they all contained Gal. However, the cell walls from some clade 1 strains also contained GalNAc, which was not present in B. anthracis cell walls. Three recently identified clade 1 strains of B. cereus that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241, had cell wall compositions that closely resembled those of the B. anthracis strains. It was also observed that B. anthracis strains cell wall glycosyl compositions differed from one another in a plasmid-dependent manner. When plasmid pXO2 was absent, the ManNAc/Gal ratio decreased, while the Glc/Gal ratio increased. Also, deletion of atxA, a global regulatory gene, from a pXO2- strain resulted in cell walls with an even greater level of Glc.

Role of different moieties from the lipooligosaccharide molecule in biological activities of the Moraxella catarrhalis outer membrane

Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS). The core OS can be dissected into a linker and three OS chains. To gain an insight into the biological activities of the LOS molecules of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35Elgt3 LOS mutant. MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo(2) (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity.

Primary Cell Wall Structure in the Evolution of Land Plants

Abstract Investigation of the primary cell walls of lower plants improves our understanding of the cell biology of these organisms but also has the potential to improve our understanding of cell wall structure and function in angiosperms that evolved from lower plants. Cell walls were prepared from eight species, ranging from a moss to advanced gymnosperms, and subjected to sequential chemical extraction to separate the main polysaccharide fractions. The glycosyl compositions of these fractions were then determined by gas chromatography. The results were compared among the eight plants and among data from related studies reported in the existing published reports to identify structural features that have been either highly conserved or clearly modified during evolution. Among the highly conserved features are the presence of a cellulose framework, the presence of certain hemicelluloses such as xyloglucan, and the presence of rhamnogalacturonan II, a domain in pectic polysaccharides. Among the modified features are the abundance of mannosyl‐containing hemicelluloses and the presence of methylated sugars.

Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan (AG)--peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several antitubercular drugs. For instance, ethambutol (EMB) targets AG biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB, as well as the single Emb from Corynebacterium glutamicum. Here, we present for the first time an experimental analysis of the membrane topology of Emb. The domain organization clearly positions highly conserved loop regions, like the recognized glycosyltransferase C motif and the hydrophilic C-terminus towards the periplasmic side of the cell. Moreover, the assignment and orientation of hydrophobic segments identified a loop region, which might dip into the membrane and could possibly line a transportation channel for the emerging substrate. Site-directed mutations introduced into plasmid-encoded Cg-emb were analyzed in a C. glutamicumDeltaemb strain for their AG glycosyl composition and linkage analysis. Mutations analyzed did not perturb galactan synthesis; however, D297A produced a dramatically reduced arabinan content and prevented growth, indicating an inactive Emb. A second D298A mutation also drastically reduced arabinan content; however, growth of the corresponding mutant was not altered, indicating a certain tolerance of this mutation in terms of Emb function. A W659L-P667A-Q674E triple mutation in the chain length regulation motif (Pro-motif) resulted in a reduced arabinose deposition in AG but retained all arabinofuranosyl linkages. Taken together, the data clearly define important residues of Emb involved in arabinan domain formation and, for the first time, shed new light on the topology of this important enzyme.

The Structure of the Major Cell Wall Polysaccharide of Bacillus anthracis Is Species-specific

In this report we describe the structure of the polysaccharide released from Bacillus anthracis vegetative cell walls by aqueous hydrogen fluoride (HF). This HF-released polysaccharide (HF-PS) was isolated and structurally characterized from the Ames, Sterne, and Pasteur strains of B. anthracis. The HF-PSs were also isolated from the closely related Bacillus cereus ATCC 10987 strain, and from the B. cereus ATCC 14579 type strain and compared with those of B. anthracis. The structure of the B. anthracis HF-PS was determined by glycosyl composition and linkage analyses, matrix-assisted laser desorption-time of flight mass spectrometry, and one- and two-dimensional nuclear magnetic resonance spectroscopy. The HF-PSs from all of the B. anthracis isolates had an identical structure consisting of an amino sugar backbone of -->6)-alpha-GlcNAc-(1-->4)-beta-ManNAc-(1-->4)-beta-GlcNAc-(1-->, in which the alpha-GlcNAc residue is substituted with alpha-Gal and beta-Gal at O-3 and O-4, respectively, and the beta-GlcNAc substituted with alpha-Gal at O-3. There is some variability in the presence of two of these three Gal substitutions. Comparison with the HF-PSs from B. cereus ATCC 10987 and B. cereus ATCC 14579 showed that the B. anthracis structure was clearly different from each of these HF-PSs and, furthermore, that the B. cereus ATCC 10987 HF-PS structure was different from that of B. cereus ATCC 14579. The presence of a B. anthracis-specific polysaccharide structure in its vegetative cell wall is discussed with regard to its relationship to those of other Bacillus species.

Structure of a capsular polysaccharide isolated from Salmonella enteritidis

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

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N-glycans has been developed. The TOF MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH(2) are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O-glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms.

Interactions between aroma compounds and whole mannoprotein isolated from Saccharomyces cerevisiae strains

The interactions of four wine aroma compounds (isoamyl acetate, hexanol, ethyl hexanoate, β-ionone) either with a whole mannoprotein extract or with mannoprotein fractions at a level encountered in wine (150 mg/L) were studied by dynamic and static headspace techniques. The mannoproteins were isolated from a synthetic medium subjected to an alcoholic fermentation with two enological yeast strains. They were fractionated by exclusion chromatography and characterized through glycosyl composition analysis. While the volatility of isoamyl acetate was affected neither by whole extracts nor by mannoprotein fractions, a significant decrease of the volatility (up to 80%) was observed for the other aroma compounds. If the behaviour of mannoproteins towards ethyl hexanoate differed between the two strains, a similar retention was found for the other compounds. The retention level was not the same when whole mannoprotein extract and isolated mannoprotein fractions were used, suggesting the possible interactions between mannoprotein fractions.

The structure of the lipopolysaccharide from a galU mutant of Pseudomonas aeruginosa serogroup-O11

The lipopolysaccharide (LPS) of a galU mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain, was sequentially extracted with phenol–chloroform–petroleum ether (PCP) followed by hot phenol–water extraction of the bacterial pellet remaining after PCP extraction. LPS was found in both the PCP extract as well as in the water phase of the hot phenol–water extract. Analysis of the carbohydrate portion released by mild acid hydrolysis of both LPS preparations, both before and after removal of all phosphate groups by treatment with aqueous HF, was performed by glycosyl composition and linkage analyses as well as by NMR and mass spectrometric analyses. The results showed that the carbohydrate portion of these two LPS extracts contained the same structure: namely, α-GalN(Ala)-(1→3)-α-(7-Cm)HepII-(1→3)-α-HepI-(1→5)-α-Kdo-(2→. The oligosaccharide preparation from PCP-extracted LPS consisted of a variety of structures containing up to six phosphate groups present as mono-, pyro-, and possibly triphosphate, primarily located on the HepI residue with some molecules having a monophosphate on HepII. The oligosaccharide preparation from the hot phenol–water-extracted LPS contained a similar variety of structures, but with an additional structure in which HepI contained a PPEA group at O-2. In addition, PAGE immunoblot analysis of the crude cellular extract with anti-A-antibodies revealed the presence of A-band material in both PA103 and the galU mutant. The A-band material was purified and characterized by glycosyl composition and linkage analyses, as well as by NMR spectroscopy, which confirmed that the A-band rhamnan polysaccharide was present but not as typical LPS since lipid-A or LPS core oligosaccharide components were not detected.

A β-(1 → 3)-arabinopyranosyltransferase that transfers a single arabinopyranose onto arabino-oligosaccharides in mung bean ( Vigna radiate) hypocotyls

Arabinopyranosyltransferase (Ara pT) activity that results in the transfer of a single arabinopyranose (Ara p) residue from UDP-β- l-arabinopyranose (UDP-Ara p) to exogenous (1 → 5)-linked α- l-arabino-oligosaccharides labeled with 2-aminobenzamide (2-AB) at their reducing ends was identified in a particulate preparation obtained from 3-day-old mung bean ( Vigna radiate L. Wilezek) hypocotyls. The transferred Ara residue was shown to be β-(1 → 3)-linked to O-3 of the non-reducing terminal Ara f residues of the oligosaccharide using nuclear magnetic resonance spectroscopy together with glycosyl composition and glycosyl linkage composition analyses. The 2AB-labeled arabino-octasaccharide was the most effective acceptor substrate analyzed, although arabino-oligosaccharides with a degree of polymerization between 4 and 7 were also acceptor substrates. Maximum Ara pT activity was obtained at pH 6.5–7.0, and 20 °C in the presence of 25 mM Mn 2+ and 0.5% Triton X-100.

Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide.

Structural and functional characterization of glycosylation in an immunoglobulin G1 to Cryptococcus neoformans glucuronoxylomannan

Analysis of the N-linked oligosaccharides of the murine IgG1 monoclonal antibody (mAb) to Cryptococcus neoformans by LC/MS revealed five different core fucosylated, biantennary complex-type oligosaccharides at Asn-293, with the major species being a mono-galactosylated oligosaccharide with the glycosyl composition of Hex 4HexNAc 4Fuc (39% of the total glycan pool). The primary sequence predicted from nucleic acid sequencing differed from that measured by mass spectrometry at position 33 (ASN to ASP), a finding that may represent post-translational modification caused by spontaneous ASP deamination. Analysis of mAb 18B7 from three hybridoma clones revealed the same heterogenous N-glycan pattern, indicating that diversity in oligosaccharide structures originated from individual cells. The binding of native and de-glycosylated mAb 18B7 to cryptococcal Ag was comparable but the de-glycosylated 18B7 had shorter serum half-life and did not activate complement (C). De-glycosylated mAb 18B7 was opsonic for C. neoformans with murine macrophages through a mechanism that involved C-independent ingestion through the C receptor. Passive administration of de-glycosylated mAb 18B7 mediated comparable protective efficacy to the native mAb in mice with lethal infection. The results imply that the contribution of N-glycan structure to immunoglobulin function varies depending on the Ag–Ab system.

The Lysine-rich Arabinogalactan-protein Subfamily in Arabidopsis: Gene Expression, Glycoprotein Purification and Biochemical Characterization

AtAGP17, AtAGP18 and AtAGP19 are homologous genes encoding three putative glycosylphosphatidylinositol (GPI)-anchored classical arabinogalactan-proteins (AGPs) in Arabidopsis. They are distinguished from other AGPs by a short, C-terminal lysine-rich region. Organ-specific expression of these genes was revealed by Northern blot analysis. AtAGP17 was strongly expressed in leaves and stems, and weakly expressed in flowers and roots; AtAGP18 was strongly expressed in flowers, and moderately expressed in roots, stems and young leaves; and AtAGP19 was strongly expressed in stems, moderately expressed in flowers and roots, and weakly expressed in young leaves. One of these genes, AtAGP17, was expressed and purified as a green fluorescent protein (GFP) fusion protein in transgenic tobacco cells using hydrophobic interaction chromatography, size exclusion chromatography and reverse phase high-performance liquid chromatography. The fusion (glyco)protein produced a characteristic AGP 'smear' with a molecular mass of 80-150 kDa when detected by Western blot analysis. Glycosyl composition and linkage analyses of purified GFP-AtAGP17 showed that carbohydrate accounted for approximately 86% of the molecule, with arabinose and galactose as major, and rhamnose and glucuronic acid as minor glycosyl residues and with 1,3,6-galactose, 1,4-glucuronic acid, 1,3-galactose and terminal arabinose as major linkages. GFP-AtAGP17 was also precipitated by beta-Yariv reagent, further confirming that AtAGP17 is a bona fide AGP. Confocal fluorescence microscopy of plasmolysed, transformed cells indicated that AtAGP17 is localized on the plasma membrane and in Hechtian strands. Hydroxyproline (Hyp) glycoside profiles of GFP-AtAGP17 in conjunction with the deduced protein sequence also served to corroborate the Hyp contiguity hypothesis, which predicts contiguous Hyp residues as attachment sites for arabinosides and clustered, non-contiguous Hyp residues as attachment sites for arabinogalactan polysaccharides.

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.

Use of Genome-Wide Expression Profiling and Mutagenesis To Study the Intestinal Lifestyle of Campylobacter jejuni

Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection, C. jejuni must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of C. jejuni was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of C. jejuni reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several C. jejuni genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that C. jejuni extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes, hspR, hrcA, spoT, Cj0571, Cj0178, Cj0341, and fliD, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by Campylobacter to resist or evade the host immune responses.

Polysaccharide production from callus cultures of Cereus peruvianus Mill. (Cactaceae)

The stems of adult plants and calli of Cereus peruvianus were used for polysaccharide extraction and characterization. The carbohydrate contents of polysaccharide extracted from stems and calli tissues were estimated to be 58.08% and 35.21%, respectively. The ratio carbohydrate contents of the plant stems and callus cells was approximately 1.65:1. The glycosyl composition of the stems and callus polysaccharides consisted mostly in galactose, arabinose, and rhamnose. This shows that calli induced from hypocotyl are capable of producing significant amounts of the same polysaccharides present in stems of adult plants.

Analysis and glycosyl composition of the exopolysaccharide isolated from the floc-forming wastewater bacterium Thauera sp. MZ1T.

Conditions were developed for the reproducible production, isolation and characterization of a novel microbial extracellular polysaccharide believed to be involved in transient viscous bulking at an industrial wastewater treatment plant. The exopolysaccharide was extracted from cell-free culture supernatants of Thauera sp. strain MZ1T grown on a minimal medium with succinate. The purified polymer was found to be approximately 260 kDa in size by gel-permeation chromatography. The GC-MS analysis of the alditol acetate and per-O-trimethylsilyl methyl glycoside derivatives revealed that the exopolysaccharide was composed of four monosaccharides including: rhamnose, galacturonic acid, N-acetylglucosamine and N-acetylfucosamine. Glucose, which also appeared at low levels, is most likely from a co-eluting glucan. The FTIR and NMR spectroscopic analyses further revealed the presence of esterified component groups on the polymer. These results represent the first published description of a polysaccharide from a member of the genus Thauera, and lay the foundation for a deeper understanding of the factors potentially involved in zoogloeal cluster formation and viscous bulking.