The crude reaction mixture was separated by TSK-40 gel-filtration chromatography, and yielded four fractions (1-4) that were all subjected
to a combination of chemical and spectroscopic analyses. Fraction 1 was established to be a mannose-reducing tetrasaccharide and contained a slight amount of a tetrasaccharide, in which galactose replaced the non reducing mannose end as follows: Fraction 2 was found to be a trisaccharide: α-D-Manp-(1→2)-α-D-Manp-(1→2)-D-Man-red, fraction 3 consisted of the disaccharide α-D-Manp-(1→2)-D-Man-red, and fraction 4 was only composed of reducing mannose. CHIR-99021 purchase Thus, the acetolysis showed that only three kinds of oligosaccharides were present, which were attached to the main polymer backbone, and that these branches were all attached to O-2 of a 2,6-disubstituted mannose. Moreover, the galactose residue, when present, was only located at the non-reducing end of a tetrasaccharide.
Thus, from both selective degradation reactions, it could be concluded that the galacto-mannan polymer is an intricate structure consisting of a 6-substituted mannan backbone with small branching chains (one to three units) of Manp residues. Furthermore, the 3-substituted mannose is only present in the trisaccharide lateral chain. The overall structure of this complex EPS is shown in Figure 5. Figure 5 Proposed structure of the EPS of H. somni 2336. When 2336 and 129Pt were grown with and without Neu5Ac added to the culture medium, only traces of Neu5Ac were present in the purified EPS of 129Pt without Neu5Ac (Figure 6, left panel), with Bortezomib mw Neu5Ac (Figure 6, right panel), or in 2336 grown without Neu5Ac (Figure 7, left panels). However, a significantly larger acetylcholine quantity of Neu5Ac was
present in the EPS of 2336 grown with Neu5Ac (Figure 7, right panels). Furthermore, the EPS also contained two additional aminosugars: N-acetylglucosamine and N-acetylgalactosamine. Figure 6 Chromatogram GC-MS of H. somni 129 pt grown without Neu5Ac (left) and with Neu5Ac (right). Figure 7 Chromatogram GC-MS of H. somni 2336 grown without Neu5Ac (top left) and with Neu5Ac (top right), and chromatogram expansion GC-MS of 2336 grown without Neu5Ac (bottom left) and with Neu5Ac (bottom right). Association of the exopolysaccharide with biofilm The presence of EPS in the H. somni biofilm was examined by cryo-ITEM following incubation of the fixed samples with antiserum to EPS and Protein-A gold particles. The Protein-A gold particles bound to the bacterial surface and in spaces between the cells, which appeared to be the residual biofilm matrix. However, no gold particles were seen in the control sample incubated without antiserum (Figure 8). Figure 8 Immuno-transmission electron micrographs of the OCT cryosection of an H. somni biofilm. H.
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