The Isolation of Two Different Lipopolysaccharide Fractions from Various Proteus mirabilis Strains

Abstract

Four distinct Proteus mirabilis strains were extracted by the phenol/water procedure. After ultracentrifugation of the dialyzed water phase, the pelleted lipopolysaccharide was purified and analyzed. The sugar composition of this lipopolysaccharide fraction I was similar for all four strains, containing only small amounts of strain-specific constituents. A second lipopolysaccharide fraction was isolated from the supernatant above (termed L1 fraction) after removal of nucleic acids. DEAE-cellulose chromatography indicated that this material is not a polysaccharide but rather a water-soluble lipopolysaccharide containing strain-specific constituents such as uronic acids, amino acids, amino sugars, neutral sugars, ethanolamine and phosphate, depending on the strain from which lipopolysaccharide I1 was isolated.Lipopolysaccharides are main constituents of the outer membrane of gramnegative bacteria. A large body of knowledge has been accumulated during the last decade concerning the chemical structure, biosynthesis and biological function of lipopolysaccharide from the Escherichia coli-Salmonella group (for review see [ 1,2]).The lipopolysaccharide from Proteus mirabilis has not been characterized very well. Kotelko and coworkers [3] reported the isolation of two different polysaccharides by acidic extraction procedures. They also showed that in a number of Proteus mirabilis strains uronic acids are constituents of the lipopolysaccharide rather than of a capsular or K-like substance [4,5]. Dmitriev et al.[6] extracted the polysaccharides from various Proteus strains with hot acetic acid and classified these strains as smooth or rough strains according to the elution pattern of the extracted polysaccharides on a Sephadex G50 column.In this paper a convenient procedure is reported for the isolation and separation of two different lipopolysaccharide fractions from each of four distinct Proteus mirabilis strains. Whereas one of these fractions resembles the lipopolysaccharide extracted from smooth strains of Salmonella, the other resembles the lipopolysaccharide extracted from rough strains of Salmonella. The smooth type lipopolysaccharide from Proteus mirabilis contains either uronic acids or phos- phate esters as 0 side chain constituents, in addition to neutral sugars and amino sugars or amino acids. MATERIALS AND METHODS Organisms and Growth ConditionsStrains D52, VI and 19 were obtained from Dr H. H. Martin (this laboratory); strain 19 Q was obtained from Dr G. Schmidt (Max-Planck-Institut fur Immunbiologie, Freiburg i. Br., Germany) and had originally been received from Dr F. Qrskov (Statens Serum Institute, Kobenhavn, Denmark). Bacteria were cultivated in complex medium as described previously [7]. Some batches were kindly grown by Dr S. Schlecht (Max-Planck-Institut fur Immunbiologie, Freiburg i. Br., Germany) [8]. Isolation of L@opolysaccharideWhole cells (20-30 g dry weight) were extracted by the hot phenollwater procedure of Westphal and Jann [9]. The water phase was dialyzed extensively against water, concen...

Structures of new acidic O‐specific polysaccharides of the bacterium Proteus mirabilis serogroups O26 and O30

“…LPS was isolated by phenol-water extraction [8] and purified by ultracentrifugation followed by digestion with nucleases [9]. After degradation of LPS with 1% HOAc at 100°C [10], O-specific polysaccharide was isolated by gel chromatography on a column (40 cm × 2.5 cm) of Sephadex G-50 in 0.05 M pyridinium acetate buffer (pH 4.5) monitored with a Knauer differential refractometer.…”

The Ribitol‐Phosphate‐Containing Lipopolysaccharide from Proteus mirabilis, Strain D52

“…Whereas the sedimenting fraction contains only small amounts of strain-specific constituents, the water-soluble fraction found in the supernatant, glucose, 8.0 % galactose, 14.5 % ribitol, 2.6 % ethanolamine and 4.3% phosphate in addition to lipid A and core-specific sugars [5]. Structural investigations on this lipopolysaccharide I1 are reported in the present paper.…”

“…Proteus mirabilis strain D52 seems to be peculiar in that its specific polysaccharide contains ribitol phosphate and ethanolamine phosphate as charged constituents [5].…”

Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness

“…SDS-PAGE of proteins in membrane fractions was carried out in a slab gel apparatus (GE4; Pharmacia LKB) according to Lugtenberg et al (1975) as previously described (Nixdorff et al, 1977). Estimation of the 0-polysaccharide chain length of LPS was made by amino acid analysis of lysine and N-acetyl-D-glucosamine as described by Gmeiner (1975). LPS was extracted from whole cells of the bacterial form and the spheroplast form by the phenol/water method (Westphal et al, 1952).…”

“…LPS from P. mirabilis VI contains, as strain-specific components, lysine and galactosamine in the O-polysaccharide chain, which can be measured by amino acid analysis (Gmeiner, 1975). Therefore, determination of the amounts of these two components per mole of 3-OH-14:O (lipid A) gives an estimation of the relative length of the 0-polysaccharide chain.…”