| Summary: | Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including <i>Aeromonas</i> spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and <sup>1</sup>H and <sup>13</sup>C NMR spectroscopy techniques were employed to study the O-PS of <i>Aeromonas hydrophila</i> strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of <i>A. hydrophila</i> JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep<sub>6</sub>Hex<sub>1</sub>HexN<sub>1</sub>HexNAc<sub>1</sub>Kdo<sub>1</sub>P<sub>1</sub>. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-<span style="font-variant: small-caps;">l</span>-mannose (<i>N</i>-acetyl-<span style="font-variant: small-caps;">l</span>-perosamine, <span style="font-variant: small-caps;">l</span>-Rha<i>p</i>4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>NAc and two α-<span style="font-variant: small-caps;">l</span>-Rha<i>p</i>4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of <span style="font-variant: small-caps;">l</span>-Rha<i>p</i>4NAc. The following structures of the O-polysaccharides were established: O-PS1 →3)-α-<span style="font-variant: small-caps;">l</span>-Rha<i>p</i>4NAc-(1→4)-α-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>NAc-(1→3)-α-<span style="font-variant: small-caps;">l</span>-Rha<i>p</i>4NAc-(1→ O-PS2 →2)-α-<span style="font-variant: small-caps;">l</span>-Rha<i>p</i>4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the <span style="font-variant: small-caps;">l</span>-configuration as a component of bacterial O-chain polysaccharides.
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