Giuseppe Impallomeni

The structure of a polysaccharide from infectious strains of Burkholderia cepacia.

The structure of an acidic exopolysaccharide (EPS) from eight strains of Burkholderia cepacia has been investigated by methylation and sugar analysis, periodate oxidation-Smith degradation, and partial acid-hydrolysis. An enzyme preparation obtained from the same organisms producing the EPS was also used to depolymerize the polysaccharide. Detailed NMR studies of the chemical and enzymatic degradation products showed that this EPS consists of a highly branched heptasaccharide-repeating unit with the following structure: [abstract: see text]. About three O-acetyl groups per repeating unit are present at undetermined positions.

Linkage analysis in disaccharides by electrospray mass spectrometry.

Analysis of the structures of complex carbohydrates requires knowledge of the identity, anomeric configuration, and sequence of the sugar residues, and identification of the reducing terminus and the positions of the glycosidic linkages. Desorption-m.s. and f.a.b.-m.s. are powerful techniques for determining the sequence, the pattern of branching, and the molecular weight of oligosaccharides containing up to 30 sugar units, and the structure of the aglycon14. Negative-ion tandem-f.a.b.-m.s. can be used to discriminate between the linkage positions in underivatised oligosaccharides5,6. Electrospray (e.s.) ionisation has also been described7-9. Although most of the applications have been concerned with the determination of molecular weights and sequencing of proteins, some studies have shown that it can be applied to carbohydrates, and we now report its application in the linkage analysis of reducing disaccharides. The negative-ion e-S.-mass spectra of (1~2)(sophorose), (l-+3)(laminaribiose), (1+4)(cellobiose), and (l-+6)(gentiobiose), /I-linked glucodisaccharides shown in Figs. l-4, respectively, reflect the positions of the linkages. Each mass spectrum contains peaks at m/z 34 1 for (M H)and at m/z I79 and 16 1 associated with fragmentation which involves the glycosidic linkages. In addition to these peaks, there are peaks at m/z 323 (sophorose, Fig. l), 28 1 (cellobiose and gentiobiose, Figs. 3 and 4), 263 (sophorose and cellobiose Figs. 1 and 3), 251 (gentiobiose, Fig. 4), and 221 (sophorose, cellobiose, and gentiobiose, Figs. 1,3, and 4). For laminaribiose, only the peaks at m/z 341, 179, and 161 are present (Fig. 2). The peak at m/z 323 is due to loss of water from the (M H)ion, and those at m/z 281, 263, 251, and 221 are associated with fragments from the sugar rings which are diagnostic of the position of the linkage. These fragmentations are likely to involve the reducing moiety, since the non-reducing moieties are identical in the four disaccharides.

Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis. A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source. The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy. On both media an exopolysaccharide having the following repeating unit was produced: -->5)-beta-Kdop-(2-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1--> This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B. pseudomallei, the microbial agent causing melioidosis. In addition to this, when grown on the mannitol-rich medium, B. cepacia strain BTS13 produced another polysaccharide that was established to be levan: -->6)-beta-D-Fruf-(2-->. The content of levan was about 20% (w/w) of the total amount of polymers. The ability of B. cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.

Metabolism of xyloglucan generates xylose-deficient oligosaccharide subunits of this polysaccharide in etiolated peas

Oligosaccharide subunits of xyloglucan were isolated from the stems and roots of etiolated pea plants and structurally characterized. The two most abundant subunits of pea xyloglucan are the well-known nonasaccharide, XXFG, and heptasaccharide, XXXG. In addition, significant amounts of oligosaccharides that have not previously been reported to be subunits of pea xyloglucan were detected, including a decasaccharide, XLFG, two octasaccharides, XLXG and XXLG, a pentasaccharide, XXG, and a trisaccharide, XG. Several novel oligosaccharide subunits, including the octasaccharide, GXFG, and the hexasaccharide, GXXG, were also found. Xyloglucan oligosaccharides generated by treatment of intact pea stem cell walls were compared to oligosaccharides generated by endoglucanase treatment of xyloglucan polysaccharides obtained by subsequent alkali extraction of the same cell walls. The results suggest that the xyloglucan in etiolated pea stems is distributed between at least two domains, one of which is distinguished by its enzyme accessibility. We further hypothesize that the chemical modification of a xyloglucan during cell-wall maturation depends on its physical environment (i.e., the domain in which it resides). For example, only the endoglucanase-released material, representing the enzyme-accessible xyloglucan domain, contains significant amounts of the two unusual oligosaccharide subunits, GXXG and GXFG, both of which have a nonreducing terminal glucosyl residue. This structure may be generated during cell-wall maturation by the sequential action of an endolytic enzyme (such as xyloglucan endotransglycosylase or endoglucanase) and an alpha-xylosidase.

Eleven newly characterized xyloglucan oligoglycosyl alditols" the specific effects of sidechain structure and location on 1H NMR chemical shifts

Eleven previously uncharacterized oligosaccharides, each containing from seventeen to twenty glycosyl residues, were isolated from the xyloglucan produced by suspension-cultured Acer pseudo-platanus cells and characterized by 1H NMR spectroscopy, fast-atom bombardment mass spectrometry, and matrix-assisted laser-desorption mass spectrometry. The complex mixture of xyloglucan oligosaccharides released by endo-(1-->4)-beta-glucanase (Trichoderma reesei) treatment of cell walls was similar to that released by digestion of the soluble xyloglucan present in the culture medium. The oligosaccharides were converted to oligoglycosyl alditols by borohydride reduction and purified by a combination of gel-permeation (Bio-Gel P-2) chromatography, normal-phase HPLC, reversed-phase HPLC, and high-performance anion-exchange (HPAE) chromatography. Eleven new oligoglycosyl alditols (along with several others that had been previously characterized) were isolated and characterized, allowing additional correlations between xyloglucan structure and specific chemical shift effects in the 1H NMR spectra to be determined. The correlations between structural and spectral features deduced in this study will facilitate the structural determination of a wide range of xyloglucans and their subunit oligosaccharides.

A new undecassacharide subunit of xyloglucans with two α-l-fucosyl residues

A new oligosaccharide subunit of xyloglucan was isolated from the beta-(1----4)-endoglucanase digestion products of the xyloglucan in what is referred to as sycamore extracellular polysaccharides and found to be an undecasaccharide having two terminal alpha-L-fucopyranosyl residues. The undecasaccharide was structurally characterized by 1H-n.m.r. spectroscopy, fast-atom bombardment mass spectrometry (f.a.b.-m.s.), and glycosyl-residue and glycosyl-linkage composition analyses. The structure of the undecasaccharide was confirmed by digesting it with a hydrolase that releases alpha-D-Xylp-(1----6)-D-Glc from the non-reducing end of xyloglucan oligosaccharides.

The structure of the exocellular polysaccharide from the cyanobacterium Cyanospira capsulata.

The exocellular polysaccharide produced by the cyanobacterium Cyanospira capsulata has been subjected to partial acid hydrolysis and N-deacetylation-nitrous acid deamination. The oligosaccharides released have been isolated by weak anion exchange and aqueous size exclusion chromatography, and characterized by a combination of 1D and 2D nuclear magnetic resonance spectroscopy, mass spectrometry, sugar composition and linkage analyses. The polysaccharide has an octasaccharide repeating unit with the following structure: [formula: see text]

O-Acetyl location on Cepacian, the principal exopolysaccharide of Burkholderia cepacia complex bacteria

Cepacian is an exopolysaccharide produced by the majority of the isolates belonging to the Burkholderia cepacia complex bacteria, a group of 17 species, some of which infect cystic fibrosis patients, sometime with fatal outcome. The repeating unit of cepacian consists of a backbone having a trisaccharidic repeating unit with three side chains, as reported in the formula below. The exopolysaccharide is also acetylated, carrying from one to three acetyl esters per repeating unit, depending on the strain examined. The consequences of O-acetyl substitution in a polysaccharide are important both for its biological functions and for industrial applications, including the preparation of conjugated vaccines, since O-acetyl groups are important immunogenic determinants. The location of acetyl groups was achieved by NMR spectroscopy and ESI mass spectrometry and revealed that these substituents are scattered in non-stoichiometric ratio on many sugar residues in different positions, a feature which adds to the already unique carbohydrate structure of the polysaccharide.

Carbon source effects on the mono/dirhamnolipid ratio produced by Pseudomonas aeruginosa L05, a new human respiratory isolate

Pseudomonas strains produce rhamnolipid mixtures (RLs) that generally consist of one or two molecules of rhamnose linked to one or two molecules of 3-hydroxyalkanoic acid. This study evaluates carbon source effects (glycerol, glucose, myristic acid, and Brassica carinata oil) on the synthesis of monorhamnolipids (mono-RLs) versus dirhamnolipids (di-RLs) in a human isolate of Pseudomonas aeruginosa PAL05. Spectrophotometry, an emulsifying index (E24) test, and an orcinol assay confirmed the production of RLs by PAL05. Purified RLs were characterized by 1H NMR analysis. PAL05 primarily produces mono-RLs when provided carbon sources containing long chain fatty acids (FAs) (myristic acid and B. carinata oil) and di-RLs when provided glycerol or glucose. qRT-PCR analysis showed that delayed expression of rhlC occurred when B. carinata oil was used, but not glycerol, glucose, or myristic acid. Our data show that the carbon source influenced the transcriptional expression of the rhlC gene and, consequently, the predominance of mono-RLs or di-RLs in PAL05 cultures.

Biosynthesis and structural characterization of polyhydroxyalkanoates produced by Pseudomonas aeruginosa ATCC 27853 from long odd-chain fatty acids

Pseudomonas aeruginosa ATCC 27853 was cultured on media containing long odd-chain fatty acids. Heptadecanoic, nonadecanoic, and heneicosanoic acids sustained cell growth and resulted in polyhydroxyalkanoate (PHA) accumulation when culturing was conducted under nitrogen starvation conditions. No PHA was produced using a complete or magnesium-deprived medium. The isolated polyesters were characterized by gas chromatography and liquid chromatography-electrospray ionization mass spectrometry (ESI-MS) of methanolyzed samples, 1H and 13C NMR spectroscopy, gel permeation chromatography, ESI MS of partially pyrolyzed samples, and differential scanning calorimetry. These PHAs are composed of seven different odd-chain repeating units starting from 3-hydroxyvalerate, with the highest species being the, to date, unreported constituent 3-hydroxyheptadecanoate, and minor amounts of 2 or 3 even-chain comonomers. The PHAs are soft, sticky, rubber-like materials having glass transition temperatures between -45 and -39°C, melting temperatures between 48 and 52°C, enthalpies of melting around 11J/g, and molar masses ranging from 77 to 188kg/mol. Statistical analysis of the ESI mass spectra of the products of their partial pyrolysis showed that they are pure copolymers and not a blend of copolymers or homopolymers.