Release of Ethanolamine Pyrophosphate during Mild Acid Hydrolysis of the Lipopolysaccharide of Pseudomonas aeruginosa

Abstract

Low molecular weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudornonas aeruginosa were isolated from the fraction containing the partially degraded polysaccharide, by successive chromatography on columns of Sephadex G-75 and G-10, followed (as necessary) by preparative high voltage paper electrophoresis. The major components identified were 2-keto-3-deoxyoctonic acid, basic amino acids (free and bound), inorganic orthophosphate, ethanolamine phosphate and ethanolamine pyrophosphate. Ethanolamine pyrophosphate has not previously been found in acid hydrolysates of lipopolysaccharides. Ethanolamine phosphate and some of the orthophosphate were apparently produced by breakdown of ethanolamine pyrophosphate.The lipopolysaccharide component of the walls of gram-negative bacteria can be readily cleaved by acid hydrolysis into polysaccharide and lipid fractions. Mild hydrolysis by lo/, acetic acid a t 100 "C for 1 to 2 h is an excellent way of achieving this with, for example, Xalmonella El], Escherichia coli [2,3] and Pseudornonas aeruginosa [4]. The water-insoluble lipid (lipid A) can be removed from hydrolysates by centrifugation or extraction into chloroform, while the partially degraded polysaccharide may be fractionated by chromatography on Sephadex. For polysaccharides from smooth bacterial strains, three fractions may be obtained [2-41. The two major fractions appear to correspond to side chain and core regions of the intact polysaccharide. Little attention has been paid to the third fraction, containing low molecular weight materials, although 2-keto-3-deoxyoctonic acid (KDO) and phosphate have been detected [2,4]. This paper presents the results obtained during a detailed study of the low molecular weight solutes released by mild acid hydrolysis of the lipopolysaccharide from P. aeruginosa NCTC 1999. MATERIALS AND METHODS Growth of Bacteria and Preparationof Lipopolysaccharide Walls were prepared by methods described previously [5] from cells of P. aeruginosa NCTC 1999 grown in nutrient broth (Oxoid No. 2) for 16 h a t Unusual Abbreviation. KDO, 2-keto-3-deoxyoctonic acid.

Elucidation of the structure of an alanine‐lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4

“…A much higher phosphate content, up to 10 or more phosphate groups, has been reported for LPS of P. aeruginosa, and most of them are considered to be located in the core region [12–14], although some phosphate may be present as triphosphate residues [13]. On mild acid hydrolysis, much of the phosphate can be released as P i , PP i and ethanolamine mono‐, di‐ and triphosphates [42,43]. No more phosphate groups could be retained in the core after alkaline degradation of the LPS of P. aeruginosa [10] as di‐ and triphosphates are unstable under alkaline conditions as well (P. M. Sánchez Carballo et al, unpublished data).…”

Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa

“…The LPS may include up to 10 phosphate groups, most of which are located in the core region. Some phosphate may occur as di‐ and tri‐phosphate residues [19,32,34] and be substituted with ethanolamine [34–36]. The LPS of P. aeruginosa 2192 studied in this work contained monophosphate and diphosphate groups, but no significant amounts of triphosphate groups or ethanolamine (data of 31 P‐ and 1 H‐NMR spectroscopy).…”

Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin

“…Earlier studies by Newton (16) demonstrated that Mg"4 and polymyxin B competed for a P. aeruginosa cellular site, which he postulated to be polyphosphate in nature. P. aeruginosa LPS has been demonstrated to have an especially high phosphate concentration (5) and may have at least 8 mol of phosphate per mol of LPS in the heptose-2-keto-3-deoxyoctonate region (A. Kropinski, personal communication). Since phosphoryl and phosphodiester groups are negatively charged at neutral pH, this high-phosphate region of the LPS would provide binding sites for Mg2", thus explaining the relatively high Mg"4 content of P. aeruginosa cell envelopes (2).…”