Lipopolysaccharide was isolated from a phage-selected mutant of a wild strain of Aeromonas salmonicida by the aqueous phenol method. The lipopolysaccharide consisted of the R form, containing per mole, three moles of L-glycero-D-manno-heptopyranose, one mole of 3-deoxy-~-manno-2-octulosonic acid (dOclA) and lipid A. The dOclA was not fully assayable by the thiobarbituric acid methods usually used, but its degradation product was detected, after Smith degradation of the lipopolysaccharide, either as free 3-deoxy-2-heptulosonic acid (after hydrolysis) or substituted by a mannopyranosyl residue derived from heptose. Mass spectrometry indicated that the dOclA existed in the furanose form and was substituted by the heptose trisaccharide through position six. Methylation analysis, chemical degradation, chromium trioxide oxidation and nuclear magnetic resonance spectroscopy were used to identify the structure of the core oligosaccharide as:Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish, and is a member of the Vibrionaceae family of bacteria, many of which are responsible for disease in fish. The position which 3-deoxy-~-manno-2-octulosonic acid (dOclA) occupies in the lipopolysaccharides of this family is not yet determined, mainly because of the difficulty experienced in analyzing for this residue. In recent years there are reports varying from the absence of dOclA in these lipopolysaccharides [l] to the presence of dOclA, but in a form in which the 5 position was phosphorylated making detection by thiobarbituric acid difficult [2] ; we have also reported the presence of 'dOclA-like' residues [3]. In our previous work on a wild strain of A. salmonicida [4] we reported a lack of dOclA in the molecule. We have now approached this problem again by phage-selecting a deep rough mutant from a wild strain of A. salmonicida in order to obtain a core oligosaccharide with a small number of sugar residues. Using this technique any dOclA residues become a much larger percentage of the total sugar residues studied, thus aiding their detection. This paper presents results which show that dOclA is indeed present, but in the rather unusual furanose form. As the furanose form is not susceptible to periodate oxidation within the ring, no malonaldehyde can be released and the chromophore with thiobarbituric acid is not formed. This is presumably the reason for the difficulty in analysing for dOclA in these strains with standard assay procedures. Abbreviations. dOclA, 3-deoxy-~-rnanno-2-octu~osonic acid ; Hep, heptose; pfu, plaque-forming units; GC/MS, gas chromatography/ mass spectrometry.
MATERIALS AND METHODS
Bacterial cultureThe parent strain 438 was isolated without the A-layer. Strain 438-R was a spontaneous phage-resistant mutant of A. salmonicida strain 438 and was isolated by spreading a mixture of lo8 bacteria and lo9 plaque-forming units (pfu) of a mixture of phage strains 37, 40RR2.8t, 54.8cL55R.1 and 59-1 on trypticase soy agar and incubating at 22°C for 48 h. The A . salmonicida phage strains...
